Bacteriophage gene 3 protein compositions and use as amyloid binding agents

ABSTRACT

The invention relates to agents and to pharmaceutical compositions for reducing the formation of amyloid and/or for promoting the disaggregation of amyloid proteins. The compositions may also be used to detect amyloid.

This is a division of application Ser. No. 14/361,157, filed May 28,2014, which is a national stage filing under 35 U.S.C. §371 ofInternational Application No. PCT/US2012/066793 filed Nov. 28, 2012, andclaims the benefit of U.S. provisional application No. 61/564,602 filedNov. 29, 2011, U.S. provisional application No. 61/708,709, Oct. 2,2012, and U.S. provisional application No. 61/730,316 filed Nov. 27,2012, all of which are incorporated herein by reference.

The invention relates to pharmaceutical compositions comprising thefilamentous bacteriophage g3p protein, amyloid-binding fragments of g3p,and amyloid-binding mutants and variants of g3p, and to the use of suchcompositions as a therapeutic to decrease amyloid load associated withdiseases, such as systemic and peripheral amyloid diseases,neurodegenerative diseases including neurodegenerative tauopathies, andtransmissible spongiform encephalopathies (prion-associated diseases).Also encompassed is the use of those compositions to prevent theaccumulation of amyloid load associated with these diseases, and the useof those compositions as diagnostics to detect amyloid and thus,diagnose such diseases.

Filamentous bacteriophage M13, and related filamentous phage, have shownutility in animal models of protein misfolding disease, and thereforerepresent a potential therapeutic class for protein misfolding diseases.See United States patent publication US 2011/0142803, incorporated byreference herein in its entirety. In particular, it has been discoveredthat filamentous bacteriophage have the ability to mediate clearance ofamyloid that have already formed in the brain. See, e.g., WO2006083795and WO2010060073, incorporated by reference herein in their entirety.

Amyloid forming diseases are characterized by neuronal degeneration andthe presence of misfolded, aggregated proteins in the brain. Thesemisfolded and aggregated proteins vary in different diseases, but inmost cases, they have a cross-beta-pleated sheet structure that bindsCongo Red dye and shows apple green birefringence. Removal of amyloid isexpected to reduce, slow the progression of, or even to reverse thesymptoms associated with a variety of diseases characterized by amyloid.

Potential therapeutic approaches to prevent and/or reverse the pathologyand/or symptoms associated with amyloid forming diseases include, forexample, inhibiting amyloid formation, promoting amyloid clearance, andinhibiting amyloid aggregation. See, for example, Aguzzi & O-Connor,Nature Review Drug Discovery (2010) 9:237-48. Removing and/or preventingthe formation of toxic oligomers may also be beneficial in the treatmentand prevention of amyloid forming diseases. Id.

Neurodegenerative diseases known to be associated with misfolded and/oraggregated proteins include Alzheimer's disease, Parkinson's disease,prion diseases, neurodegenerative tauopathies, amyotrophic lateralsclerosis (ALS), spinocerebellar ataxia (SCA1), (SCA3), (SCA6), (SCA7),Huntington disease, entatorubral-pallidoluysian atrophy, spinal andbulbar muscular atrophy, hereditary cerebral amyloid angiopathy,familial amyloidosis, frontotemporal lobar degeneration (FTLD) includingfrontotemporal lobe dementia, British/Danish dementia, and familialencephalopathy. Other diseases involve misfolded and/or aggregatedproteins in the periphery—the so called peripheral amyloidoses. See, forexample, Chiti & Dobson, Annu Rev Biochem (2006) 75:333-66; and Josephset al., Acta Neuropathol (2011) 122:137-153. There is a great need toprevent and/or reduce amyloid aggregate formation (i.e., misfoldedand/or aggregated proteins) to treat or reduce the symptoms or severityof these diseases.

Recently, the National Institute on Aging and the Alzheimer'sAssociation published criteria for diagnosing “all-cause” andAlzheimer's Disease dementia. See, McKhann et al., Alzheimer's &Dementia, (2011) 7(3):263-9. Based on this guidance, “all cause”dementia is diagnosed when behavioral or cognitive symptoms satisfy fivetests, which include, for example, the interference with the ability tofunction at work or at usual activities, and a decline from previouslevels of functioning and performing. The tests involve a combination ofhistory-taking and objective cognitive assessment. As described herein,the discovery that filamentous bacteriophage g3p protein,amyloid-binding fragments of g3p, and amyloid-binding mutants andvariants of g3p bind amyloid provides complementary methods to diagnoseany disease or dementia resulting from the formation of amyloid,including “all cause” and Alzheimer's dementia.

Filamentous bacteriophage are a group of structurally related virusesthat infect bacterial cells, and contain a circular single-stranded DNAgenome. They do not kill their host during productive infection. Raschedand Oberer, Microbiol Rev (1986) 50:401-427. Examples of filamentousbacteriophage include phage of the Ff family (e.g., M13, f1, and fd).The nucleotide sequence of fd has been known since 1978. Beck et al.,Nucleic Acids Research (1978) 5(12):4495-4503. The full sequence of M13was published in 1980. van Wezenbeek et al., Gene (1980) 11:129-148.Phage f1 was sequenced by 1982. Hill and Petersen, J. Virol. (1982)44(1):32-46. The f1 genome comprises 6407 nucleotides, one less thanphage fd. It differs from the fd sequence by 186 nucleotides (includingone nucleotide deletion), leading to 12 amino acid differences betweenthe proteins of phages f1 and fd. The f1 sequence differs from that ofM13 by 52 nucleotides, resulting in 5 amino acid differences between thecorresponding proteins. Id. The DNA sequences of M13 and fd vary at 192(3%) nucleotides, yet only 12 of these differences result in a change inthe corresponding amino acid sequence (6.25%). van Wezenbeek et al.,Gene (1980) 11:129-148.

The structure of filamentous phage is well established and is reviewed,for example, in Marvin, Curr. Opin. in Struct. Biol. (1998) 8:150-158;Rasched and Oberer, Microbiological Reviews (1986) 50(4):401-427.Filamentous phage have a “coat” that comprises thousands of copies of amajor capsid protein encoded by gene 8 (g8p, p8 or pVIII). It is theassembled g8p-DNA complex that forms the characteristic filamentousshape of the phage. Minor coat proteins, i.e., those that are present inonly a few (3-5) copies, are located at the ends of the filament. One ofthese tip proteins, g3p (also known as p3 or pIII), is necessary forbacterial host binding and initiates infection.

M13 phage has a mature g3p of 406 amino acids. GenBank Ref SeqNP_(—)510891.1 provides a reference sequence that includes the 18residue amino-terminal signal sequence. Variants that have amino aciddifferences as compared to published sequences are common. Filamentousphage of the I-family have g3p that differs from Ff family members, buteven between families g3p is still highly conserved. Stassen et al., JMol Evol (1992) 34:141-52.

A crystal structure is available for g3p. Lubkowski et al., Structure(1998) 7(6) 711-722. The protein comprises 3 folded domains separated byflexible glycine-rich linker sequences. There are two amino-terminaldomains, N1 and N2 comprising 262 amino acids, that interact to form anN1-N2 complex. The carboxy-terminal (CT, also called N3) domain is 146amino acids and it serves to anchor g3p in the phage particle byhydrophobic interactions with g8p. Marvin, Current Opin. in StructuralBiology (1998) 8:150-158. A publically available ribbon structureprepared using the N1-N2 domain fusion protein 2g3p of Holliger, J Mol.Biol. (1999) 288(4):649-57 is presented in FIG. 1.

Unlike most proteins, unfolding of the N1 and N2 domains from the latent“locked” form is required for g3p to acquire its native biologicalactivity. Eckert & Schmid, J. Mol. Biol. (2007) 373:452-461. In theinitial step in infection, N2 binds the bacterial F-pilus via residueson the outer rim of N2. Deng & Perham, 2002. This initial binding by N2“unlocks” g3p by “opening” the N1-N2 complex, permitting N1 to then bindthe co-receptor ToIA. In an N1-N2 fragment of g3p, the thermaltransition for the initial unlocking step in which N2 unfolds occurs ata melting temperature (T_(M)) of 48.1° C. Part of the process involvesan isomerization at the GIn212-Pro213 peptide bond. Pro213 converting istrans in the unlocked state. N1 remains stably folded until the secondstep, which occurs at a T_(M) of 60.2° C. Reviewed in Eckert & Schmid,2007.

Mutations in the N1-N2 fragment have been used to study the stabilityand infectivity of various mutants. Eckert & Schmid, 2007. One variant,designated “3A” impaired pilus binding and decreased the stability ofthe N2 domain. For this mutation, the T_(M) is decreased to 42.6° C. 3Acarries the following mutations: W181A, F190A, and F194A. Another mutantin N2, G153D, destabilized N2, decreasing T_(M) to 44.4° C. A Q129Hmutant stabilized N2, increasing the T_(M) to 51.4° C. The IY variantcontains the mutations T101I and D209Y in the hinge and increases thestability of the N1-N2 fragment (T_(M)=56.5° C.). IHY contains themutations T101I, Q129H, and D209Y (T_(M)=60.1° C.). IIHY contains themutations T13I, T101I, Q129H, and D209Y (T_(M)=61.8° C.). Both the Q129Yand T13I mutations are stabilizing, and adding these mutations furtherincreases the melting temperature, T_(M). Phage infectivity variedinversely with the strength of the domain interactions within g3p.Eckert & Schmid, 2007. Deletion of the N2 domain (phage fd(ΔN2))increased the infectivity by removing the blocking effect of the N2domain on N1-binding of ToIA. Id.

The invention is based in part on the discovery that g3p also mediatesbinding of the filamentous phage to amyloid in a manner analogous to theprocess by which phage infect bacteria. U.S. Pat. No. 7,867,487postulated that the mechanism underlying the therapeutic efficacy ofphage in disaggregating amyloid reported in that patent was that thephage's long, thin, structure, might enable it to organize along theamyloid fibers. In addition, it was proposed that the high alpha helicalcontent present in g8p, the major coat protein, might interfere with thebeta sheet structure of amyloid. That mechanism is consistent with thepatent's report that nanomolar amounts of phage can disaggregatemicromolar quantities of β-amyloid, which would suggest a high copycomponent of the phage, i.e., g8p, is mediating the effect. It is alsoconsistent with the report in US20110182948 that tobacco mosaic virus,which has a similar structure to filamentous phage, can causedisaggregation. Thus this earlier work suggested that either the intactstructure (a long filament with many alpha helices) was important fortherapeutic effect or that, if a particular coat protein was important,it was a protein that was highly represented on the phage coat, such asg8p. None of this earlier work provided any suggestion that an isolatedcomponent of bacteriophage, as opposed to intact phage, could bind toamyloid and/or cause its disaggregation. Moreover, there was never anysuggestion that a minor coat protein of filamentous bacteriophage playeda role in its ability to bind to and disaggregate amyloid.

However, this disclosure provides evidence of an alternative (althoughnot necessarily mutually exclusive) mechanism of action. The inventorhas found that phage g3p directly binds amyloid fibers and thatphage-mediated disaggregation is dependent upon this initial bindingstep. The inventor's recognition that g3p is responsible for filamentousphage-mediated amyloid binding provides a mechanism for bacteriophagetherapeutic efficacy, as well as provides a basis for new classes oftherapeutics and diagnostics.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a ribbon structure of the N1 and N2 domains of g3p, andthe hinge.

FIGS. 2A-2C present alignments of g3p's from different sources. FIG. 2Ais an alignment of g3p from phage M13 (SEQ ID NO: 1), Fd (SEQ ID NO:2),and F1 (SEQ ID NO: 3), including a consensus sequence (SEQ ID NO: 4).FIG. 2B shows an alignment of g3p from phage I2-2 (SEQ ID NO: 5) and Ike(SEQ ID NO: 6), along with a consensus sequence between I2-2 and Ike(SEQ ID NO: 7). FIG. 2C presents the amino acid sequence of g3p fromphage If (SEQ ID NO: 8).

FIG. 3A presents a surface plasmon resonance (SPR) study of phagebinding. Binding to Aβ fibrils was compared to binding to Aβ monomersusing 10¹⁴ phage/mL flowed across the biosensor chip. FIG. 3B shows theK_(a), K_(d), and K_(D) calculated from the SPR data shown in FIG. 3A.

FIGS. 4A and 4B present binding studies. FIG. 4A shows a direct bindingassay for two phage doses (10¹¹/mL and 10¹²/mL) with increasing molaramounts of fAβ42. FIG. 4B is a binding competition study and provides analternate way to determine the K_(D) for M13 binding. Construct 1 wasused.

FIG. 5 shows binding competition results using heat denatured (boxes−90° C. for 10 minutes) versus native conformation (circles) M13(Construct 1) in the amyloid fiber binding competition assay.

FIG. 6 shows a Thioflavin T (ThT) fluorescence assay using fAβ42incubated in the presence or absence of 2 concentrations of M13 phage(Construct 1).

FIGS. 7A and 7B show the effect of varying individual assay parametersin the ThT disaggregation assay. FIG. 7A presents disaggregationpercentages in the presence of two salt concentrations (0.15 M and 1.5M). FIG. 7B presents percentages of fAβ remaining at two temperatures(4° C. and 37° C.). Construct 1 was used.

FIGS. 8A and 8B represent Mβ-amyloid binding assays using fAβ42. In FIG.8A, M13 binding is reported using incubation temperatures from 18° C. to58° C. for 3 hours. FIG. 8B shows binding kinetics for incubations at37° C. vs. 50° C.

FIGS. 9A-9C show the effect of proteolytic removal of g3p onphage-amyloid interactions. The protease Arg C was used to clip g3p fromM13 phage (M13Δg3p). FIG. 9A presents the results of an Aβ bindingcompetition study using M13Δg3p phage compared to native (treatedidentically to the ArgC-treated phage but without protease treatment)phage. FIG. 9B shows the effect of Arg C treatment on infectivity of theM13Δg3p phage compared to native phage. FIG. 9C compares ArgC treatedphage to native phage in the disaggregation assay.

FIGS. 10A and 10B present the results of a binding competition assayusing a N1-N2 fragment of g3p, herein referred to as recombinant solubleN1N2 (rs-g3p(N1N2); “Construct 3”), M13Δg3p (Arg C treated), and M13 ascompetitors of labeled M13 binding to fAβ42. FIG. 10B shows a repeat ofthe competition assay.

FIG. 11 presents competition data for phage fd, IIHY, AAA, and M13.Phages fd, AAA, and IIHY were pre-activated at 50° C. for 1.5 hours,then activated and non-activated Fd, AAA, & IIHY were compared for theirability to compete with labeled M13 for binding to Aβ during a 45 minuteincubation at 37° C.

FIG. 12A shows a schematic of rs-g3p(N1N2) (Construct 3). FIG. 12Bpresents an ion exchange profile for rs-g3p(N1N2). FIG. 12C shows theresults of a gel filtration assay using Sephacryl S-300 andrs-g3p(N1N2). FIG. 12D shows a Western Blot of rs-g3p(N1N2) togetherwith g3p and g8p controls. M13 phage are run in lanes 1 and 2 as apositive control, and detected with a polyclonal anti-M13 antibody,which detects both g8p and g3p. Purified rs-g3p is run in lanes 3 and 4,and detected with the same polyclonal anti-M13 antibody.

FIG. 13 presents SPR data using rs-g3p(N1N2) (Construct 3). rs-g3p(N1N2)potently binds fAβ42 with a K_(D) of about 160 nM, but does not bindmonomers.

FIG. 14A and FIG. 14B present a ThT fluorescence assay used to measurethe amyloid present in a given sample. 10 μM of Aβ42 monomers wasincubated in the presence or absence of 5 concentrations of rs-g3p(N1N2)(Construct 3) at 37° C. for 3 days. The amount of fibers formed at theend of 3 days as measured by quantitating the bound ThT fluorescence.The IC₅₀ is approximately 20 nM indicating that rs-g3p(N1N2) potentlyinhibits formation of Aβ42 fibers (FIG. 14B). FIG. 14A indicates thatbinding is dose-dependant.

FIG. 15A shows the transmission electron micrography (TEM) results ofincubating fAβ42 in the presence or absence of rs-g3p(N1N2) (Construct3). FIG. 15B shows the results of a ThT fluorescence assay using Aβ42and 2 μM rs-g3p(N1N2) (Construct 3) incubated at 37° C. for 7 days.rs-g3p(N1N2) blocks the formation of fAβ42.

FIG. 16 demonstrates that rs-g3p(N1N2) (Construct 3) potently inhibitsthe formation of α-synuclein fibers. 25 μM of α-synuclein was assembledby agitating at 300 rpm for 4 days at 37° C. (see, Bar 1). The secondbar on the graph represents alpha-synuclein monomers plus 1×10⁻¹³pentameric M13 phage shaking at 37° C. for 3 days. The results shown inbar 2 indicate that pentameric M13 blocks assembly of α-synucleinfibers. The third bar on the graph represents alpha-synuclein monomers+83 nM rsg3p monomers. The results shown in bar 3 indicate that monomersare less effective at inhibiting α-synuclein fiber formation thanpentameric M13. Bar 4 is a negative control showing alpha synucleinmonomers at time zero. In bar 5, g3p monomers without α-synuclein fibersis shown to determine whether g3p binds to pTAA and sequesters the dyefrom binding to the fibers. The results shown in bar 5 indicate that g3pdoes not bind to pTAA.

FIG. 17 presents competition binding data for rs-g3p(N1N2) (Construct3), M13 (Construct 2), rs-g3p(N1N2)-hIgG4-Fc fusion protein (Construct4), and an IgG4-Fc negative control.

FIG. 18 presents competition binding data comparing M13 (Construct 2;squares), rs-g3p(N1N2) (Construct 3; triangles), rs-g3p(N1N2)-hIgG4-Fcfusion protein (Construct 4; upside down triangles), and a recombinantIgG4-Fc negative control (diamonds).

FIG. 19 shows a filter trap assay comparing five concentrations of Aβ42fibers plus or minus two concentrations of M13 (Construct 2), 800 nMrs-g3p(N1N2) (Construct 3), and three concentrations ofrs-g3p(N1N2)-hIgG4-Fc fusion protein (Construct 4).

FIG. 20 presents competition binding data for rs-g3p(N1N2) (Construct 3;“monomer”) and streptavidin conjugated rs-g3p(N1N2)(“SA[g3pN1N2]_(n=2-4)”; “SA-g3p”; “tetramer”). rs-g3p(N1N2) and SA-g3pwere compared for their ability to compete with labeled M13 for bindingto Aβ during a three hour incubation at 37° C.

FIG. 21 shows a filter trap assay comparing five concentrations of fAβ42plus or minus two concentrations of rs-g3p(N1N2) (Construct 3;“monomer”) and two concentrations of SA-g3p (“tetramer”).

FIGS. 22A and 22B show TEMs of fAβ42 at times zero (FIG. 22A) and threedays after incubation with SA-g3p (FIG. 22B).

FIG. 23 shows the amino acid sequence of one rs-g3p(N1N2)-hIgG4-Fcconstruct “Construct 4” (SEQ ID NO:9). The N1N2 region of “Construct 4”is derived from the N1N2 region of “Construct 1” (SEQ ID NO:10).

FIG. 24 shows the amino acid sequence of another rs-g3p(N1N2)-hIgG4-Fcconstruct “Construct 5” (SEQ ID NO:11). The N1N2 region of “Construct 5”is derived from the N1N2 region of “Construct 2” (SEQ ID NO:12).

FIG. 25 shows the amino acid sequence of one rs-g3p(N1N2)-hIgG1-Fcconstruct “Construct 6” (SEQ ID NO:13). The N1N2 region of “Construct 6”is derived from the N1N2 region of “Construct 2”.

FIG. 26 shows the amino acid sequence alignment of N2 from: fd (SEQ IDNO:14), f1 (SEQ ID NO:15), M13 (SEQ ID NO:16), Ike (SEQ ID NO:17), 12-2(SEQ ID NO:18), and If1 (SEQ ID NO:19). An asterisk “*” indicatespositions which have a single, fully conserved residue. A colon “:”indicates conservation between groups of strongly similar propertiesthat score greater than 0.5 in the Gonnet PAM 250 matrix. A period “.”indicates conservation between groups of weakly similar properties thatscore equal to or less than 0.5 in the Gonnet PAM 250 matrix.

FIG. 27A shows a schematic of Construct 5. FIG. 27B shows the DNAsequence of the g3p portion of Construct 5 (SEQ ID NO:23). FIG. 27Cshows the amino acid sequence of the g3p portion of Construct 5 (SEQ IDNO:24).

FIG. 28 shows the results of an experiment testing two rs-g3p(N1N2)-IgGfusion proteins for their ability to reduce amyloid β in a transgenicmouse model of Alzheimer's Disease. rs-g3p(N1N2)-hIgG4-Fc (Construct 5)and rs-g3p(N1N2)-hIgG1-Fc (Construct 6) both significantly reduced thelevel of amyloid β in the hippocampus of Alzheimer's Diseased mice.

FIG. 29 shows the results of an experiment testing two rs-g3p(N1N2)-IgGfusion proteins for their ability to reduce amyloid β in a transgenicmouse model of Alzheimer's Disease. rs-g3p(N1N2)-hIgG4-Fc (Construct 5)and rs-g3p(N1N2)-hIgG1-Fc (Construct 6) were both able to significantlyreduce the level of amyloid β in the cerebral cortex of Alzheimer'sDisease mice.

FIG. 30A and FIG. 30B show assembly inhibition of Aβ42 withrs-g3p(N1N2)-hIgG1-Fc (Construct 6). FIG. 30A shows a “native” agarosegel made without SDS. The samples were run in TEA buffer without SDS andnot boiled. The results indicate that Construct 6 is capable ofinhibiting the assembly of fAβ42. FIG. 30B presents a Tht fluorescenceassay used to measure the amyloid present in a given sample. 10 μM ofAβ42 monomers were incubated in the presence or absence of 2concentrations of rs-g3p(N1N2)-hIgG1-Fc (Construct 6) at 37° C. for 1day. The amount of fibers formed at the end of day 1 was measured byquantitating the bound ThT fluorescence. rs-g3p(N1N2)-hIgG1-Fc(Construct 6) potently inhibits formation of Aβ42 fibers. The figurealso indicates that inhibition of fiber formation with Construct 6 isdose-dependent.

FIG. 31A, FIG. 31B, FIG. 31C, and FIG. 31D present representativecircular dichroism data showing that Aβ42 assembly is inhibited byrs-g3p(N1N2) (Construct 3). Circular dichrosism measures the α-helix andβ-sheet content of the Aβ fibers to be assessed. FIG. 31A shows theellipticity versus wavelength for Aβ42 at T=0, T=24 hours, and T=48hours. FIG. 31B shows ellipticity versus wavelength for Aβ42 plusConstruct 3 at T=0, T=24 hours, and T=48 hours. FIG. 31C shows arepresentative ThT assay where the amount of fibers formed between 24and 48 hours was measured by quantitating the bound ThT fluorescence.Construct 3 potently inhibits formation of Aβ42 fibers. FIG. 31D showsellipticity versus wavelength for Construct 3 at T=0, T=24 hours, andT=48 hours. Taken together, these data confirm the ability of Construct3 to inhibit Aβ42 assembly.

FIG. 32 presents representative data showing that M13 (Construct 2) andrs-g3p(N1N2)-hIgG1-Fc (Construct 6) block oligomer-induced toxicity ofN2a cells. See, e.g., Stine et al. (2003) J. Biol. Chem. 278(13):11612-11622 and Stine et al. (2011) Erik D. Roberson (ed.) Alzheimer'sDisease and Frontotemporal Dementia, Methods in Molecular Biology, vol.670: 13-32. N2a cells were differentiated by serum starvation for 48hours prior to treatment. Aβ42 oligomers (2 uM) were pre-incubated withConstruct 2 and Construct 6 at 37° C. for 3 hrs before addition to N2acells. Time zero (“TO”) complexes were not pre-incubated. After 24 hoursof incubation, adenylate kinase (“AK”) release was monitored. AK releaseinto the medium indicates cell death/lysis. Aβ42 oligomers were made asdescribed by Stine et. al., 2011. The results indicate that M13 andrs-g3p(N1N2)-hIgG1-Fc are potent inhibitors of toxic oligomers.

FIG. 33 shows a filter trap assay comparing six concentrations of Aβ42fibers plus or minus 1×10¹²/ml M13 (Construct 2); 80 nm and 800 nMrs-g3p(N1N2)-hIgG4-Fc construct (Construct 5); and 80 nm and 800 nM ofrs-g3p(N1N2)-hIgG1-Fc (Construct 6). Aβ42 fibers were incubated withConstructs 2, 5, and 6 at 37° C. for 3 days, followed by filterretardation. The filter was probed by mAb 6E10 (1:15000), whichrecognizes Aβ42 fibers trapped on the filter. 800 nM of Construct 5 orConstruct 6 equals 5×10¹⁴/ml Construct 2 by molecular molarity. Theresults indicate that Constructs 2, 5, and 6 potently disaggregateβ-amyloid fibers.

FIGS. 34A and 34B present representative assays used to measure theamount of M13 (Construct 2) bound to fAβ42 after 3 hours ofpreincubation with ftau. 5 μM of Aβ42 monomers bound to Construct 2 wasincubated in the presence or absence of 4 concentrations of ftau at 37°C. for 3 hours. Since fAbeta:M13-Alexa488 pellets but ftau:M13-Alexa488does not pellet, measuring the loss of fluorescence from the pelletedmaterial indicates that ftau competed the fAbeta binding. Here, theamount of M13-fAβ formed at the end of 3 hours was measured byquantitating the Alexa488 fluorescence in the pelleted bindingcompetition reaction. The results indicate that ftau is able to competewith M13-Alexa488 (Construct 2) for fAβ42 binding.

FIG. 35 shows the results of one representative SPR assay testing theability of rs-g3p(N1N2)-hIgG4-Fc (Construct 4) to bind to ftau. Theresults indicate that Construct 4 potently binds ftau.

FIG. 36A and FIG. 36B show the ability of rs-g3p(N1N2)-hIgG1-Fc(Construct 6) to disaggregate ftau. Tau fibers were prepared by diluting40 uM of the microtubule binding repeat region (“MTBR”) of tau into 50mM superoxide dismutase (“Sod”). Various concentrations of Construct 6and the prepared ftau were incubated in acetate buffer at pH7.0, 37° C.for 72 hrs. ThT fluorescence was recorded in the presence of 5 foldexcess ThT. FIG. 36A presents the results of a representative ThT assayshowing the ability of Construct 6 to disaggregate ftau. FIG. 36B showsanother representative experiment confirming the ability of Construct 6to disaggregate tau. FIGS. 36A and 36B also show that disaggregation offtau by Construct 6 is dose dependent.

FIG. 37A, FIG. 37B, FIG. 37C, and FIG. 37D present representativeexperiments showing the inhibition of Aβ aggregation byrs-g3p(N1N2)-hIgG1-Fc (Construct 6) and rs-g3p(N1N2) (Construct 3) overtime. Aβ42 was dissolved in DMSO and diluted into PBS containing NaN3.Aβ42 was aggregated at 37° C. plus or minus various concentrations ofConstruct 3 and Construct 6. Aggregation of Aβ42 was measured by ThTfluorescence. FIG. 37A shows an SDS PAGE of the samples. FIG. 37B showsthe results from one representative experiment. FIG. 37C shows theresults from another representative experiment. FIG. 37D summarizes theresults.

FIG. 38A and FIG. 38B present the results of experiments showing theability of rs-g3p(N1N2)-hIgG1-Fc (Construct 6) to block the conversionof PrP to PrP—Sc. Construct 6 and IgG cell lysates were subjected toultra-centrifugation to separate soluble (supernatant) and insoluble(pellet) PrP species. PrP species were visualized biochemically with ananti-PrP monoclonal antibody (6D11). In the presence of IgG, there is apartitioning of PrP in both soluble and insoluble fractions. In thepresence of Construct 6, there is limited insoluble PrP. Data representsn=4.

FIG. 39A and FIG. 39B present the results of experiments showing theability of rs-g3p(N1N2)-hIgG1-Fc (Construct 6) to reduce theaccumulation and aggregation of PrP^(Sc) in a cell culture model ofprion disease. FIG. 39A shows biochemically resolved undigested andPK-digested N2a22L^(Sc) cell lysates following treatment with Construct6 and IgG. A significant reduction in PrP^(Sc) levels is clearlyobserved in cells treated with increasing concentrations of Construct 6.An approximately 50% reduction in PrP^(Sc) levels is achieved withtreatment of ˜0.08 ug/ml Construct 6. Treatment with 10 ug/ml Construct6 reduces PrP^(Sc) levels to 5.725%, p<0.0001. No marked changes inPrP^(Sc) levels were observed in N2A22L^(Sc) cells treated with 1 ug/mlmurine IgG. For FIG. 39B, the X-ray films were subsequently digitizedand initially normalized to the effect in IgG treated N2a22L^(Sc) cellsfrom the same passage which was considered to be 100%. The densitometrydata from PK-digested blots was then analyzed relative to theequivalently blotted undigested lysates and expressed as a percentchange PrP^(Sc)/PrPc. Data represents n=4.

DESCRIPTION OF EMBODIMENTS

The invention is based, in part, on the inventor's recognition of therole of the gene 3 protein (“g3p,” also known as “p3” or “pIII”) inmediating amyloid binding and disaggregation of amyloid aggregates. Theinvention is also based on the inventors' identification of a minimalsequence of g3p required for binding to amyloid.

Thus, in certain embodiments, the invention provides molecules, inparticular polypeptides, that comprise minimal consensus amyloid bindingsequences derived from g3p. In one aspect of these embodiments, themolecules are soluble. In another aspect of these embodiments, themolecules disaggregate and/or prevent the aggregation of amyloid (e.g.,amyloid plaque). In another aspect of these embodiments, the moleculesare fusion proteins. In a more specific aspect of these embodiments, themolecules are fusion proteins additionally comprising an amino acidsequence of an immunoglobulin chain. In an even more specific aspect ofthese embodiments, the molecules are fusion proteins additionallycomprising an amino acid sequence of an immunoglobulin G (e.g., IgG) orimmunoglobulin M (e.g., IgM) chain. In still another aspect of theseembodiments, the molecule comprises the N2 domain of g3p. In a morespecific aspect of these embodiments, the molecule comprises the N1-N2domain of g3p. In still another aspect of these embodiments, themolecule comprises a full length g3p. In yet another aspect, themolecule is a polypeptide that is a fragment, mutant, or variant of anyof the foregoing.

In other aspects, the invention provides molecules that bind to ToIA,such as ToIA inhibitor molecules, in particular polypeptides, thatcomprise minimal consensus amyloid binding sequences. The ToIA bindingmolecules and/or ToIA inhibitors of the present invention bind to,depolymerize, prevent the aggregation of, and disaggregate amyloid. TheToIA binding molecules and/or ToIA inhibitor molecules include fusionproteins. In certain embodiments the ToIA binding molecule and/or ToIAinhibitor molecule is a colicin or amyloid binding fragment of acolicin. In certain embodiments the colicin is a Group A colicin. See,e.g., Cascales et al., Microbiol. Mol. Biol. Rev. (2007) 71(1): 158-229.The ToIA binding molecules and ToIA inhibitor molecules of the inventionare useful therapeutics to decrease amyloid load associated withdiseases, such as systemic and peripheral amyloid diseases,neurodegenerative diseases including neurodegenerative tauopathies, andtransmissible spongiform encephalopathies (prion-associated diseases).Also encompassed is the use of those compositions to prevent theaccumulation of amyloid load associated with these diseases, and the useof those compositions as diagnostics to detect amyloid and thus,diagnose such diseases.

In another embodiment, the invention provides filamentous bacteriophagethat have been modified to overexpress g3p as compared to wild typephage, to express an amyloid binding fragment of g3p, an amyloid bindingmutant or variant form of g3p, or an amyloid binding fusion proteincomprising g3p.

The invention provides compositions of matter and/or pharmaceuticalcompositions of any of the foregoing molecules or bacteriophage, as wellas their use to bind to, disaggregate, and prevent aggregation ofamyloid, and to their use to detect amyloid deposits and diagnosediseases and disorders characterized by amyloid.

DEFINITIONS

The term “g3p” when used alone or in terms such as “g3p-derived” refersto any wild type or recombinant filamentous phage g3p protein (includingfragments, variants, and mutants of g3p). The term should not beconstrued as limited to any particular filamentous bacteriophage g3p. Byway of example, the term “g3p” includes SEQ ID NO: 1 and the relatedproteins shown in FIG. 2.

The term “filamentous bacteriophage” includes both wild type filamentousbacteriophage, and recombinant filamentous bacteriophage. In the presentapplication, “filamentous bacteriophage” may also be referred to as“bacteriophage,” “phage,” or “M13.”

The term “wild-type filamentous bacteriophage” as used herein refers tofilamentous phage found in nature, filamentous phage that have beenindicated as “wild-type” in any nucleotide or amino acid sequencedatabase, filamentous bacteriophage that are commercially available andcharacterized as “wild-type,” and filamentous bacteriophage that haveacquired non-recombinant mutations relative to any of the foregoingthrough passaging.

The term “domain” means a region of a polypeptide (including proteins)having some distinctive physical feature or role including for examplean independently folded structure composed of one section of apolypeptide chain. A domain may contain the sequence of the distinctivephysical feature of the polypeptide or it may contain a fragment of thephysical feature which retains its binding characteristics (i.e., it canbind to a second domain). A domain may be associated with anotherdomain. In other words, a first domain may naturally bind to a seconddomain. For example, the g3p N2 domain binds F-pili and the g3p N1domain binds ToIA.

The terms “amyloid,” “amyloid fibrils,” and “amyloid fibers,” as usedherein are generic terms for a tertiary structure that is formed byaggregation of any of several different proteins and that consists of anordered arrangement of β sheets stacked perpendicular to a fiber axis.Sunde et al., J. Mol. Biol. (1997) 273:729-39. One exemplary amyloid isthe aggregate of amyloid-β formed in Alzheimer's disease, which iscomposed of beta-amyloid peptide “βA,” which are 39-43 amino acidinternal fragments cleaved from the human amyloid precursor protein(hAPP). There are short forms, such as Aβ40, and long forms, such as themore fibrillogenic Aβ isoform, Aβ42. Other exemplary amyloid proteinsinclude misfolded α-synuclein (associated with Parkinson's disease),huntingtin (associated with Huntington's disease), tau (associated withAlzheimer's Disease), and the abnormal conformation of the prionprotein, PrP^(Sc). Additional examples are provided throughout thedescription and are known to those of skill in the art (see, e.g.,Aguzzi (2010), and Eichner and Radford, Mol. Cell (2011) 43:8-18). Thus,unless a protein or peptide is specified, use of the terms “amyloid,”“amyloid fibrils,” or “amyloid fibers” should not be construed aslimited to any particular protein or disease.

The term “beta amyloid peptide” is synonymous with “β-amyloid peptide,”“βAP,” “βA,” and “Aβ.” All of these terms refer to an amyloid formingpeptide derived from the human amyloid precursor protein (hAPP).

A phage, protein, fusion protein, fusion protein domain, or a mutant,fragment, or variant of the foregoing that “binds amyloid fibrils” orthat is “amyloid-binding” is one that is positive in an amyloid bindingassay. Amyloid binding can be detected in vitro using a direct bindingassay such as surface plasmon resonance (SPR), in which case it willgenerally bind amyloid with a Kd of at least 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, or10⁻¹¹ M. Alternatively, amyloid binding can be detected using the fAβ42binding assay described in the examples. Amyloid-binding fragments,variants, and mutants of g3p may also be identified by theirco-localization to amyloid when injected into a transgenic mouse modelof any a protein misfolding disease.

Any of the products or compositions of the invention described as“disaggregating” or “mediating disaggregation” reduce aggregates thathave already formed. Disaggregation can be measured by the filter trapassay. Wanker et al., Methods Enzymol (1999) 309:375-86. The filter trapassay is described herein and can be used both to detect aggregates andto monitor disaggregation mediated by compositions of the invention.Disaggregation is detected as decreased retention of amyloid on thefilter, as shown by a decrease in staining, in the presence ofincreasing concentrations of the disaggregating agent.

As used herein, a composition that “reduces amyloid” does one or more ofthe following: inhibits amyloid formation, causes amyloiddisaggregation, promotes amyloid clearance, inhibits amyloidaggregation, blocks and/or prevents the formation of toxic amyloidoligomers, and/or promotes the clearance of toxic amyloid oligomers.

Any of the products or compositions of the invention described as“protecting neurons from amyloid damage” prevent the accumulation of newamyloid and/or prevent the formation of toxic amyloid oligomers.Products or compositions of the invention described as “protectingneurons from amyloid damage” may be taken prophylactically. Whether ornot a product or composition protects neurons from amyloid damage may bemeasured by the neuronal cell culture cytotoxicity assay describedherein.

As used herein, “PrP protein,” “PrP,” and “prion,” refer to polypeptidesthat are capable under appropriate conditions, of inducing the formationof aggregates responsible for protein misfolding diseases. For example,normal cellular prion protein (PrP^(c)) is converted under suchconditions into the corresponding scrapie isoform (PrP^(Sc)) which isresponsible for diseases such as, but not limited to, bovine spongiformencephalopathy (BSE), or mad cow disease, feline spongiformencephalopathy of cats, kuru, Creutzfeldt-Jakob Disease (CJD),Gerstmann-Straussler-Scheinker disease (GSS), and fatal familialinsomnia (FFI).

The term “variant” as used herein in conjunction with a bacteriophage,protein, polypeptide or amino acid sequence (e.g., a g3p variant or avariant of an amyloid binding fragment of g3p), refers to acorresponding substance that contains at least one amino acid difference(substitution, insertion or deletion) as compared to the referencesubstance. In certain embodiments a “variant” has high amino acidsequence homology and/or conservative amino acid substitutions,deletions and/or insertions as compared to the reference sequence. Insome embodiments, a variant has no more than 75, 50, 40, 30, 25, 20, 15,12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acid differences as compared tothe reference sequence. A “conservative substitution” refers to thereplacement of a first amino acid by a second amino acid that does notsubstantially alter the chemical, physical and/or functional propertiesof the g3p protein or amyloid binding fragment of g3p (e.g., the g3pprotein or amyloid binding fragment retains the same charge, structure,polarity, hydrophobicity/hydrophilicity, and/or preserves functions suchas the ability to recognize, bind to, and/or reduce amyloid). Suchconservative amino acid modifications are based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplaryconservative substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine, andisoleucine.

The term “mutant” (e.g., “mutant g3p” or “mutant amyloid bindingfragment”) refers to a protein that is mutated at one or more aminoacids in order to modulate its therapeutic or diagnostic efficacy. Incertain embodiments, a mutant contains a substitution, deletion and/orinsertion at an amino that is known to interact with amyloid. In otherembodiments, a mutant contains a substitution, deletion and/or insertionat an amino that is a conserved amino acid present in a wild-type g3p oramyloid binding fragment thereof. In some embodiments, a mutant has nomore than 75, 50, 40, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1amino acid differences as compared to the reference sequence. In someembodiments, the amino acid substitutions are conservativesubstitutions. The terms “variant” and “mutant” are used interchangeablyherein except that a “variant” is typically non-recombinant in nature,whereas a “mutant” is typically recombinant.

The term “high stringency,” as used herein, includes conditions readilydetermined by the skilled artisan based on, for example, the length ofthe DNA. Generally, such conditions are defined in Sambrook et al.Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104,Cold Spring Harbor Laboratory Press (1989), and include use of aprewashing solution for the nitrocellulose filters 5×SSC, 0.5% SDS, 1.0mM EDTA (PH 8.0), hybridization conditions of 50% formamide, 6×SSC at42° C. (or other similar hybridization solution, such as Stark'ssolution, in 50% formamide at 42° C.), and with washing at approximately68° C., 0.2×SSC, 0.1% SDS. The skilled artisan will recognize that thetemperature and wash solution salt concentration can be adjusted asnecessary according to factors such as the length of the probe.

The term “moderate stringency,” as used herein, includes conditions thatcan be readily determined by those having ordinary skill in the artbased on, for example, the length of the DNA. The basic conditions areset forth by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2ded. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press (1989),and include use of a prewashing solution for the nitrocellulose filters5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of 50%formamide, 6×SSC at 42° C. (or other similar hybridization solution,such as Stark's solution, in 50% formamide at 42° C.), and washingconditions of 60° C., 0.5×SSC, 0.1% SDS.

The term “high sequence homology” means at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% amino acid sequence homology with thereference sequence as measured using known computer programs, such asthe Bestfit program.

A “fusion protein” is a non-naturally occurring protein comprising atleast two polypeptide domains.

A “g3p fusion protein” comprises a g3p protein linked to a seconddomain.

An “N1-N2 fusion protein” (also termed “N1N2 fusion protein”) comprisesthe N1 and N2 domains (or mutants, fragments, or variants of either),but not the N3/CT domain, of a g3p protein linked to a second domain. AN1N2 fusion protein may or may not comprise the hinge region.

An “N2 fusion protein” comprises the N2 domain (or mutants, fragments,or variants of N2), but neither the N1 nor N3/CT domains, of a g3pprotein linked to a second domain. A N2 fusion protein may or may notcomprise the hinge region.

As used herein, “Construct 1” is derived from wild type M13 (see,Genbank file: NC_(—)003287.2, version GI:56718463. In Construct 1, ascompared to wild type M13, Ser378(AGC) is changed to Gly(GGC), and Ile87(ATT) is changed to Asn(AAC)). Construct 1 comprises the amino acids ofSEQ ID NO: 10.

“Construct 2” is a wild type M13 isolate (GenBank JX412914.1). Construct2 comprises the amino acids of SEQ ID NO:12.

“Construct 3” is a recombinant soluble g3p fragment comprising the N1and N2 domains of g3p (rs-g3p(N1N2)) comprising the amino acids of SEQID NO:20.

“Construct 4” is recombinant soluble g3p fragment IgG4 Fc fusion protein(rs-g3p(N1N2)-hIgG4-Fc) comprising the amino acids of SEQ ID NO:9. TheN1N2 region of “Construct 4” is derived from the N1N2 region of“Construct 1.”

“Construct 5” is a recombinant soluble g3p fragment IgG4 Fc fusionprotein (rs-g3p(N1N2)-hIgG4-Fc) comprising the amino acids of SEQ IDNO:11. The N1N2 region of “Construct 5” is derived from the N1N2 regionof “Construct 2.”

“Construct 6” is recombinant soluble g3p fragment IgG1 Fc fusion protein(rs-g3p(N1N2)-hIgG1-Fc) comprising the amino acids of SEQ ID NO:13. TheN1N2 region of “Construct 6” is derived from the N1N2 region of“Construct 2.”

Sources of g3p

Filamentous bacteriophage are a group of related viruses that infectgram negative bacteria, such as, e.g., E. coli. See, e.g., Rasched andOberer, Microbiology Reviews (1986) December:401-427. Examples offilamentous bacteriophage include, but are not limited to, phage of theFf family (i.e., at least M13, f1, and fd) and phage of the 1-family(i.e., at least I22, Ike, If1).

All naturally occurring filamentous bacteriophage contain g3p as a minorcoat protein present in 3 to 5 copies per phage. Thus, in one aspect ofthe invention, an isolated g3p is obtained from any naturally occurringfilamentous bacteriophage. Recombinant forms of g3p can also beproduced. Recombinant g3p may correspond to a wild type g3p from anynaturally occurring filamentous bacteriophage. Thus, recombinant,isolated g3p is also encompassed by the invention.

One example of g3p, from phage M13, is presented in SEQ ID NO: 1. Unlessotherwise clearly specified, any g3p mutations described are inreference to SEQ ID NO: 1 shown below in clean and annotated form:

1 AETVESCLAK PHTENSFTNV WKDDKTLDRY ANYEGCLWNA TGVVVCTGDE TQCYGTWVPI 61GLAIPENEGG GSEGGGSEGG GSEGGGTKPP EYGDTPIPGY TYINPLDGTY PPGTEQNPAN 121PNPSLEESQP LNTFMFQNNR FRNRQGALTV YTGTVTQGTD PVKTYYQYTP VSSKAMYDAY 181WNGKFRDCAF HSGFNEDPFV CEYQGQSSDL PQPPVNAGGG SGGGSGGGSE GGGSEGGGSE 241GGGSEGGGSG GGSGSGDFDY EKMANANKGA MTENADENAL QSDAKGKLDS VATDYGAAID 301GFIGDVSGLA NGNGATGDFA GSNSQMAQVG DGDNSPLMNN FRQYLPSLPQ SVECRPFVFS 361AGKPYEFSID CDKINLFRGV FAFLLYVATF MYVFSTFANI LRNKES AnnotatedSEQ ID NO: 1 1AETVESCLAK PHTENSFTNV WKDDKTLDRY ANYEGCLWNA TGVVVCTGDE TQCYGTWVPI 61GLAIPENEGG GSEGGG SEGG GSEGGGTKPP EYGDTPIPGY TYINPLDGTY PPGTEQNPAN 121PNPSLEESQP LNTFMFQNNR FRNRQGALTV YTGTVTQGTD PVKTYYQYTP VSSKAMYDAY 181WNGKFRDCAF HSGFNEDPFV CEYQGQSSDL PQPPVNA GGG SGGGSGGGSE GGGSEGGGSE 241GGGSEGGGSG GGSGSG DFDY EKMANANKGA MTENADENAL QSDAKGKLDS VATDYGAAID 301GFIGDVSGLA NGNGATGDFA GSNSQMAQVG DGDNSPLMNN FRQYLPSLPQ SVECRPFVFS 361AGKPYEFSID CDKINLFRGV FAFLLYVATF MYVFSTFANI LRNKES

TABLE 1 Key for annotated SEQ ID NO: 1 Residue when signal RegionResidues peptide is present Key N1  1-67 19-85 underline G1 68-86 86-104 highlight N2  87-217 105-235 underline and bold G2 218-256236-274 italic N3 257-406 275-424 highlight and underlineSEQ ID NO: 1 is GenBank NP-510891.1 with the 18 amino acid signalpeptide removed, thus the amino acid numbering is for the mature g3p.The signal peptide is generally included in any expression construct,and immature g3p that includes the signal peptide is included within thescope of the various embodiments of the invention unless context makesclear that it is expressly excluded. SEQ ID NO: 1 is provided as areference sequence only. It is in no way intended to limit theinvention.

Sequences of g3p from multiple sources are known. Exemplary g3p aminoacid sequences for bacteriophage of the Ff family include thosesequences found in UniProt accession numbers P69169 (phage f1), P03661(phage fd), and P69168 (phage m13). Exemplary g3p amino acid sequencesfor bacteriophage of the 1-family include P15415 (phage I22), P03663(phage Ike), and 080297 (phage If1). Alignments of several g3p sequencesare presented in FIG. 2.

G3p useful in this invention also includes fragments, mutants, and/orvariants of g3p. Mutants or variants may be described with reference toa full length g3p or with reference to a fragment of g3p. Any fulllength or fragment g3p, including mutants and/or variants thereof thatretain the ability to bind to amyloid, regardless of their ability todisaggregate amyloid is within the scope of the present invention. Anyprotein “comprising” such g3p is also encompassed by the presentinvention. Likewise, proteins “including,” “consisting of,” “consistingessentially of,” or “having” such g3p are also encompassed.

Amyloid-Binding and Amyloid Disaggregating Fragments of g3p

As mentioned, g3p has two amino-terminal domains, N1 and N2, thatinteract to form an N1-N2 complex, and one carboxy-terminal domain, N3(also called “CT”). In Ff phage, the N1 domain comprises residues 1-67and the N2 domain comprises residues 87-217 of mature g3p. Residues87-123 form the hinge that allows opening and closing between N1 and N2.Sometimes the hinge is considered part of N2, whereas in other instancesit is treated as a separate element. N1 and N2 are also linked byflexible glycine-rich linker sequence. Within N1, there are twodisulphide bridges between Cys7 and Cys36 and between Cys46 and Cys53.There is a single disulphide bridge in N2 between Cys188 and Cys201. TheN3/CT domain comprises residues 257 to 406. Hollinger, 1999; Marvin,1998. In the carboxy terminal domain there is a disulphide bridgebetween Cys354 and Cys371. Marvin, 1998. There are no interdomaindisulphide bridges in g3p.

Non-limiting examples of amyloid binding fragments of g3p include the N2domain either with the hinge (e.g., at least residues 87-217 of SEQ IDNO: 1) or without the hinge (e.g., at least residues 124-217 of SEQ IDNO: 1); and the N1-N2 domains (e.g., at least residues 1-67 and 87-217of SEQ ID NO: 1), either with or without the intervening linker sequence(e.g., with or without residues 68-86 of SEQ ID NO: 1), and either withor without the hinge. In any of the foregoing examples, the N2 or N1N2fragments may be the N2 or N1N2 found in a wild-type filamentousbacteriophage or a recombinant N2 or N1N2. In any of the foregoingexamples, the N2 or N1N2 fragments may mutants or variants of thewild-type filamentous bacteriophage sequence.

Useful amyloid binding fragments of g3p include any fragment of g3p,including N2 and N1N2 fragments that retain the ability to bind toamyloid, regardless of the fragment's ability to disaggregate amyloid.Any protein “comprising” such amyloid binding fragment (or mutant orvariant thereof) is encompassed by the present invention. Likewise,proteins “including,” “consisting of,” “consisting essentially of,” or“having” the g3p fragment or variant are also encompassed.

N2 and N2 Polypeptide Mutants and Variants

A primary structure alignment of N2 from: fd, f1, M13, Ike, I2-2, andIf1 is shown as FIG. 26. The amino acids of fd are shown in SEQ IDNO:14; f1 in SEQ ID NO:15; M13 in SEQ ID NO:16; Ike in SEQ ID NO:17;12-2 in SEQ ID NO:18; and If1 in SEQ ID NO:19. Using this figure andalignment as guidance, one embodiment of the invention encompasses a N2polypeptide, N2 polypeptide mutants, and N2 polypeptide variants,comprising the amino acids of SEQ ID NO: 14, 15, 16, 17, 18, or 19,including any amyloid binding fragments thereof.

In other embodiments, the N2 polypeptide is a N2 polypeptide mutant orvariant that retains its ability to bind to amyloid and has an aminoacid sequence that comprises no more than 75, 50, 40, 30, 25, 20, 15,12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acid differences when alignedwith the amino acid sequence of any one of SEQ ID NO: 14, 15, 16, 17,18, or 19. In FIG. 26, an asterisk “*” indicates positions which have asingle, fully conserved residue. A colon “:” indicates conservationbetween groups of strongly similar properties that score greater than0.5 in the Gonnet PAM 250 matrix. A period “.” indicates conservationbetween groups of weakly similar properties that score equal to or lessthan 0.5 in the Gonnet PAM 250 matrix. In some aspects of theseembodiments, the N2 polypeptide mutant or variant does not comprise anamino acid difference at any position indicated with an “*” in FIG. 26.In more specific aspects, the N2 polypeptide mutant or variant does notcomprise an amino acid difference at any position indicated with an “*”and comprises the same amino acid as at least one of SEQ ID NO: 14, 15,16, 17, 18, or 19 at each position indicated with a “:” in FIG. 26. Ineven more specific aspects, the N2 polypeptide mutant or variant doesnot comprise an amino acid difference at any position indicated with an“*” and comprises the same amino acid as at least one of SEQ ID NO: 14,15, 16, 17, 18, or 19 at each position indicated with a “:” and eachposition indicated with a “.” in FIG. 26.

In other embodiments, a N2 polypeptide variant is described byspecifying a percent amino acid similarity to SEQ ID NO: 14, 15, 16, 17,18, or 19 again with the caveat that the N2 polypeptide variant bindsamyloid. In these embodiments, the N2 polypeptide shares at least 70%,at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity over the amino acids of reference sequences shown inSEQ ID NO: 14, 15, 16, 17, 18, or 19.

In other embodiments, a N2 polypeptide is described by specifying apercent amino acid similarity to the N2 region of SEQ ID NO: 1, againwith the caveat that the N2 polypeptide variant binds amyloid. In theseembodiments, the N2 polypeptide variant shares at least 70%, at least80%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identity over N2 region of SEQ ID NO: 1.

In still other embodiments, a N2 polypeptide is described by secondaryor tertiary structure. It is known that fd-N2 and If1-N2 domains usehomologous parts of their surfaces to bind to the same site on theF-pilus in E. coli. Lorenz et al., J Mol Biol. 405:989-1003 (2011) at,for example, 990. The amino acid residues and secondary and tertiarystructure that mediate N2 binding to F-pilus also mediate N2 binding toamyloid. Thus, amino acid residues and secondary and tertiary structuresthat are critical for N2 to F-pilus binding are also critical forN2-amyloid binding. N2 polypeptide variants comprising the amino acidsrequired to maintain the secondary and tertiary structure in the regionof N2-F-pilus binding are within the scope of the present invention.

N1N2 and N1N2 Polypeptide Mutants and Variants

A primary structure alignment of fd, f1, and M13 is shown as FIG. 2A,and Ike, I2-2, and If1 as FIG. 2B. Using this alignment as guidance, oneembodiment of the invention encompasses a N1N2 polypeptide, polypeptidemutant, or polypeptide variant comprising the amino acids that areconserved between fd, f1, and M13 or as between I2-2, Ide, and If1, asidentified with reference to the sequences of FIG. 2. In otherembodiments, the N1N2 polypeptide is a N1N2 polypeptide mutant orvariant, that retains its ability to bind to amyloid and has an aminoacid sequence that comprises no more than 75, 50, 40, 30, 25, 20, 15,12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acid differences when alignedwith the amino acid sequence of any one of SEQ ID NO: 1, 2, 3, 5 or 6.

In other embodiments, a N1N2 polypeptide, mutant, or variant isdescribed by specifying a percent amino acid similarity to the N1N2region of SEQ ID NO: 1, again with the caveat that the N1N2 polypeptidevariant binds amyloid. In these embodiments, the N1N2 polypeptidevariant shares at least 70%, at least 80%, at least 85%, at least 86%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity over N1 and N2 regionsof SEQ ID NO: 1.

Fusion Proteins

In one aspect, the invention relates to fusion proteins. The fusionprotein comprises g3p, an amyloid-binding fragment of g3p, a ToIAbinding molecule, or a ToIA inhibitor. Fusion proteins comprising mutantor variant g3p or g3p fragments are fully encompassed. The fusionprotein is linked, fused, conjugated, coupled, or associated with/to atleast one additional protein or protein domain with which it is notnormally associated. In one embodiment, the fusion protein is a g3pfusion protein that comprises a g3p protein linked to a second domain.In another embodiment, the fusion protein is an amyloid-binding fragmentof a g3p protein linked to a second domain. In another embodiment, thefusion protein is an N1N2 fusion protein, which comprises the N1 and N2domains, but not the CT domain, of a g3p protein. In still anotherembodiment, the fusion protein is an N2 fusion protein, which comprisesthe N2 domain, but neither the N1 nor CT domains, of a g3p protein. Asnoted, some aspects of the invention relate to mutated or variated g3pprotein or amyloid-binding fragments thereof, and to mutated or variatedN1N2 or N2 domains that bind amyloid fibers. Thus, fusion proteinscomprising these mutated or variated forms are also part of theinvention.

The g3p or amyloid binding fragment and the fusion partner polypeptidemay be part of a continuous amino acid sequence with the fusion partnerpolypeptide linked directly or through a short peptide linker to eitherthe N terminus or the C terminus of the g3p or amyloid binding fragmentpolypeptide. In such cases, the g3p or amyloid binding fragment thereofand the fusion partner polypeptide may be translated as a singlepolypeptide from a coding sequence that encodes both the g3p or amyloidbinding fragment thereof and the fusion partner polypeptide.

In some embodiments, the fusion protein comprises an immunoglobulinconstant region as the second domain. Fusion proteins comprised ofimmunoglobulin constant regions linked to a protein of interest, orfragment thereof, have been described (see, e.g., U.S. Pat. Nos.5,480,981 and 5,808,029; Gascoigne et al. 1987, Proc. Natl. Acad. Sci.USA 84:2936; Capon et al. 1989, Nature 337:525; Traunecker et al. 1989,Nature 339:68; Zettmeissl et al. 1990, DNA Cell Biol. USA 9:347; Byrn etal. 1990, Nature 344:667; Watson et al. 1990, J. Cell. Biol. 110:2221;Watson et al. 1991, Nature 349:164; Aruffo et al. 1990, Cell 61:1303;Linsley et al. 1991, J. Exp. Med. 173:721; Linsley et al. 1991, J. Exp.Med. 174:561; Stamenkovic et al., 1991, Cell 66:1133; Ashkenazi et al.1991, Proc. Natl. Acad. Sci. USA 88:10535; Lesslauer et al. 1991, Eur.J. Immunol. 27:2883; Peppel et al. 1991, J. Exp. Med. 174:1483; Bennettet al. 1991, J. Biol. Chem. 266:23060; Kurschner et al. 1992, J. Biol.Chem. 267:9354; Chalupny et al. 1992, Proc. Natl. Acad. Sci. USA89:10360; Ridgway and Gorman, 1991, J. Cell. Biol. 115, Abstract No.1448; Zheng et al. 1995, J. Immun. 154:5590). These molecules usuallypossess both the biological activity associated with the linked moleculeof interest as well as the effector function, or some other desiredcharacteristic associated with the immunoglobulin constant region (e.g.,biological stability, cellular secretion).

In some embodiments, the fusion protein comprises an Fc fragment of animmunoglobulin constant region. Fc expression cassettes may be purchasedcommercially. The Fc fragment can be comprised of the CH2 and CH3domains of an immunoglobulin and the hinge region of the immunoglobulin.The Fc fragment can be the Fc fragment of an IgG1, an IgG2, an IgG3 oran IgG4. In one specific embodiment, the portion of an immunoglobulinconstant region is an Fc fragment of an IgG1. In another embodiment, theportion of an immunoglobulin constant region is an Fc fragment of anIgG4. In still another embodiment, the portion of an immunoglobulinconstant region is an Fc fragment of an IgM.

Thus, in one embodiment, a recombinant soluble g3p or amyloid-bindingfragment is fused to an immunoglobulin Fc domain using standardmolecular biology techniques. The recombinant soluble g3p oramyloid-binding fragment may be mutated or variated. For example, anamyloid-binding fragment of g3p, such as the N1N2 domain or the N2domain, can be cloned into an IgGFc fusion expression vector. ExemplaryIgGFc fusion vectors include, for example, one of the pFUSE-Fc vectorsavailable from InvivoGen. In some embodiments, the resulting bivalent(e.g., g3p(N1N2)-IgGFc or g3p(N2)-IgGFc fusion protein will have higheravidity for amyloid binding than the recombinant soluble g3p since it isnow bivalent.

In other embodiments, the fusion protein comprises a non-Fc proteinlinked to a g3p or amyloid binding fragment of g3p.

In other embodiments, the fusion protein comprises at least two g3ppolypeptides or amyloid-binding fragments thereof. In other embodiments,the fusion protein comprises three or more g3p polypeptides, oramyloid-binding fragments thereof. In other embodiments, the fusionprotein comprises five g3p polypeptides, or amyloid-binding fragmentsthereof. Such dimeric and multimeric fusion proteins provide higheravidity interactions since they include more than one g3p, oramyloid-binding fragments thereof.

In other embodiments, the fusion protein comprises albumin. See forexample, U.S. Pat. No. 6,686,179 to Fleer.

In all instances, the g3p or amyloid binding fragment of g3p in thefusion protein encompasses mutants and variants thereof.

In general, the fusion proteins bind to amyloid at least as effectivelyas the corresponding unlinked g3p or g3p fragment thereof. Whenapplicable, the fusion proteins are at least as effective in mediatingdisaggregation of amyloid, promoting amyloid clearance, inhibitingamyloid aggregation, and/or removing or preventing the formation oftoxic oligomers as the corresponding unlinked g3p or fragment thereof.In some embodiments, the fusion protein binds amyloid and is at least aseffective in mediating disaggregation of amyloid, promoting amyloidclearance, inhibiting amyloid aggregation, and/or removing or preventingthe formation of toxic oligomers as is a recombinant, soluble g3pcomprising SEQ ID NO: 1. In still other embodiments, the fusion proteinbinds amyloid and is at least as effective in mediating disaggregationof amyloid, promoting amyloid clearance, inhibiting amyloid aggregation,and/or removing or preventing the formation of toxic oligomers as phageM13. In yet other embodiments, the fusion protein binds amyloid and ismore effective in mediating disaggregation of amyloid, promoting amyloidclearance, inhibiting amyloid aggregation, and/or removing or preventingthe formation of toxic oligomers than phage M13. In some embodiments,the fusion protein binds amyloid and is at least as effective inreducing amyloid in a protein misfolding disease as phage M13. In stillother embodiments, the fusion protein binds amyloid and is moreeffective in reducing amyloid in a protein misfolding disease as phageM13. In still other embodiments, the fusion protein binds amyloid and isat least or more effective in preventing amyloid formation as phage M13.

Fusion proteins can be synthesized using techniques well known in theart. For example, the fusion proteins of the invention can besynthesized recombinantly in cells (see, e.g., Sambrook et al. 1989,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y. and Ausubel et al. 1989, Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, N.Y.).Alternatively, the fusion proteins of the invention can be synthesizedusing known synthetic methods such as solid phase synthesis. Synthetictechniques are well known in the art (see, e.g., Merrifield, 1973,Chemical Polypeptides, (Katsoyannis and Panayotis eds.) pp. 335-61;Merrifield 1963, J. Am. Chem. Soc. 85:2149; Davis et al. 1985, Biochem.Intl. 10:394; Finn et al. 1976, The Proteins (3d ed.) 2:105; Erikson etal. 1976, The Proteins (3d ed.) 2:257; U.S. Pat. No. 3,941,763.Alternatively, the final construct may share essentially the samefunction as a recombinantly produced fusion protein, but simply beproduced using non-recombinant techniques, such as ligation chemistry.Components of the fusion proteins may be prepared using the same generalmethodology described for g3p expression and g3p mutations.

In some embodiment, the g3p or amyloid binding fragment (or mutant orvariant form thereof) may be fused to a marker sequence, such as apeptide that facilitates purification of the fused polypeptide (eitheralone or in addition to fusion to another protein or incorporation of acarrier molecule). The marker amino acid sequence may be ahexa-histidine peptide such as the tag provided in a pQE vector (Qiagen,Mississauga, Ontario, Canada), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. (1989) 86:821-824, for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the hemagglutinin (HA) tag, corresponds to anepitope derived from the influenza HA protein. (Wilson et al., (1984)Cell 37:767).

Phage Overexpressing g3p

In another aspect, the invention relates to bacteriophage modified toincrease the number of copies of g3p expressed by the phage to more thanthe 3-5 copies typically found in wild type filamentous bacteriophage.In one embodiment, phage that express increased numbers of g3p may beselected from naturally occurring variants. In another embodiment,recombinant techniques are used to increase the copy number of g3p.

In some embodiments, a wild type sequence encoding g3p or amyloidbinding fragments of g3p (including mutants or variants thereof) can beused to replace one of the genes encoding another bacteriophage coatprotein. Depending upon the bacteriophage gene replaced, the number ofg3p can be increased to 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, 500, 1000, or even nearly 3000 copies (for example, if the gene 3coding sequence were used to replace the gene 8 coding sequence or werefused to the end of the gene 8 coding sequence).

To produce phage expressing additional copies of g3p, a g3p codingsequence (or mutant or variant form thereof) is cloned as describedelsewhere in the description. The g3p coding sequence (or mutant orvariant form thereof) may then be used to replace another phage gene andexpressed, if necessary in conjunction with helper phage.

Alternatively, in some embodiments the g3p coding sequence (or mutant orvariant form thereof) is fused in frame to the coding sequence ofanother phage gene, either with or without an intervening “spacer”sequence. Methods of preparing phage proteins to which another proteinor peptide is “fused” are well known in the phage display art, and g3por an amyloid binding fragment thereof can be “displayed” in the samemanner as, for example, antigens or antibody chains. E.g. Scott & Smith,Science (1990) 249:386-90; Devlin et al., Science (1990) 249:404-06.When expression of only a fragment of g3p is desired, the codingsequence for the fragment (or mutant or variant form thereof) may belinked to the other gene so that one of the natural Ser/Gly linkersequences present in g3p serves as the linker. In some embodiments, onlycoding sequence for N2 or N1N2 domains (or mutant or variant formthereof) is fused in frame to the other gene.

Mutant G3P and Amyloid Binding Fragments

In another aspect, the invention relates to mutant g3p proteins andmutant amyloid-binding fragments thereof. Fusion proteins and phagecomprising the mutant g3p proteins and amyloid-binding fragments arealso part of the invention. Mutant g3p and mutant amyloid-bindingfragments thereof may be produced, or selected, for properties thatcontribute to the therapeutic efficacy of the pharmaceuticalcompositions described in this application. For example, g3p oramyloid-binding fragments thereof may be recombinantly mutated orotherwise selected to posses one or more of the following propertiesrelative to g3p of M13: increased affinity for amyloid binding, areduced hinge T_(M), increased avidity (avidity being distinguished fromaffinity in that avidity is used to describe the sum of all availableamyloid binding where a g3p comprises more than one amyloid bindingsite), increased ability to disaggregate amyloid aggregates, orincreased ability to prevent aggregation of amyloid fibrils.Alternatively, or in addition, the mutant g3p or mutant amyloidfragments thereof may incorporate other useful properties describedelsewhere in the description.

Mutant g3p proteins can be produced by mutagenesis of phage, or byrecombinant techniques, such as PCR-based site directed mutagenesis orrandom mutagenesis.

In some embodiments, mutants with higher affinity are produced bymutagenizing M13 and then selecting phage on an amyloid affinity columncoupled with stringent washing conditions. Successive rounds of binding,washing, elution, and then expansion of selected phage enriches forthose phage with high affinity binding to amyloid. Once increasedaffinity of the phage population is achieved using amyloid panning,individual clones with high affinity are selected and analyzed. In thisway, phage mutants may be selected for high affinity binding followingrandom mutagenesis.

G3p, or any amyloid binding fragments thereof, (e.g., N1N2 domains or N2domains) may also be mutagenized using recombinant techniques. Forexample, a vector as described herein carrying g3p or an amyloid bindingfragment thereof (e.g., N1N2 or N2) may be mutated using PCR-basedmutagenesis strategies. The encoded, mutated protein is then expressedand amyloid binding and affinity of the mutants assessed as described.

Mutant amyloid binding fragments of g3p may also be derived from mutantg3p. For example, by mutating g3p and/or selecting for a mutated g3pwith desirable properties and then obtaining the desired amyloid bindingfragment therefrom, e.g., by proteolysis and subsequent purification.

Screening of phage bearing mutant g3p for increased affinity of amyloidbinding, changes in temperature-sensitivity of binding, etc., may beused to identify phage for further characterization of the g3p of thatphage. Screening for properties such as temperature sensitivity ofbinding can utilize an amyloid affinity column with one or more of thebinding, washing, or elution steps conducted in a temperature dependentfashion.

In some embodiments, the mutant g3p or g3p amyloid-binding fragmentbinds amyloid with an affinity that is at least 3, 5, 10, 20, 30, 40,50, 100, 200, 300, 400, 500 or even 1000 higher than binding of thecorresponding unmutated g3p or g3p fragment from M13. In otherembodiments, the mutant g3p or g3p amyloid-binding fragment retainsamyloid-binding that is at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, or 99% as strong as binding of the corresponding unmutatedg3p or amyloid-binding g3p fragment from M13. In some embodiments amutant g3p or amyloid binding fragment that displays loweramyloid-binding affinity than the corresponding unmutated form alsopossesses another desirable biological (e.g., greater ability todisaggregate amyloid; greater ability to prevent aggregation of amyloidfibrils) or pharmaceutical (e.g., greater metabolic stability, favorablepharmacokinetic profile, greater solubility) property that is improvedas compared to the corresponding unmutated form. Amyloid binding may beassessed by surface plasmon resonance or in a competitive ELISA, asdescribed in the Examples.

In some embodiments, variants and/or mutants of g3p may be identified byscreening DNA libraries using hybridization to M13 g3p to select relatedDNAs that hybridize to M13 g3p under either high stringency or moderatestringency conditions.

In some embodiments, a mutated g3p is a recombinantly produced g3p oramyloid-binding fragment thereof that differs from mature M13 g3pprotein (SEQ ID NO: 1) by at least one amino acid residue but stillbinds amyloid. In some embodiments, individual point mutations arespecified by providing the amino acid of the M13 g3p at a particularresidue of the mature protein and the replacement amino acid at thatresidue. For example, “F194A” means the phenylalanine at position 194 ofthe mature M13 sequence has been changed to an alanine. In otherembodiments, a mutated g3p is described by specifying a percent aminoacid similarity to SEQ ID NO: 1, again with the caveat that the mutatedg3p binds amyloid fibrils. In these embodiments, the mutated g3p sharesat least 70%, at least 80%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity over the full length of SEQ IDNO: 1. In those embodiments involving a mutated amyloid binding fragmentof g3p, the mutated amyloid-binding fragment shares at least 70%, atleast 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity over the full length of the corresponding fragment ofSEQ ID NO: 1.

As a practical matter, whether any particular polypeptide is at least70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to SEQ ID NO: 1 can be determinedconventionally using known computer programs, such the Bestfit program.When using Bestfit or other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, that the percentage of identity is calculated overthe full length of the portion of the reference amino acid sequence thatis homologous to the query sequence.

In some embodiments of the various aspects, mutant g3p and amyloidbinding fragments thereof include no mutations at an amino acid residuethat is conserved among g3p of the Ff family, the 1-family, or both theFf and I-families. In other embodiments, the mutant g3p and amyloidbinding fragments thereof include at most mutations at 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acid residues that are conserved among g3p of theFf family, the 1-family, or both the Ff and I-families. In still otherembodiments, the mutant g3p and amyloid binding fragments thereofinclude at most mutations at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid residues that are not conserved among g3p of the Ff family, the1-family, or both the Ff and 1-families. In still another embodiment,the mutant g3p and amyloid binding fragments thereof include at mostmutations at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residuesthat are not conserved between one or more of I22, Ike, and If1. In yetother embodiments, the mutant g3p and amyloid binding fragments thereofinclude at most mutations at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidresidues that are not conserved among g3p of the Ff family, the1-family, or both the Ff and 1-families. In some embodiments, the atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations are located within theN1 domain. In some embodiments, the at most 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 mutations are located within the N2 domain. In some embodiments,the at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations are locatedwithin the N2 domain and are not within the hinge region.

Site directed mutagenesis may target residues known to be important forstability of g3p, N1N2, or the N2 domain. For example, alaninereplacement mutations at D94 and T95; E115; N122; L125; E126 and E127;E127 and E128; Q129; Q145; T154 and T156; Q157; T159 and D160; K163 andT164; Y166; and E196 and D197 have been previously shown to notsignificantly affect phage binding to F-pili, Deng & Perham, 2002.Accordingly, these positions are tolerant of mutation and a mutation atone or more of these positions may either enhance or have a neutraleffect on the amyloid-binding ability in the g3p and g3p amyloid-bindingfragments of the invention. Thus, in some embodiments, the inventionincludes a g3p or g3p amyloid-binding fragment that is mutated at one ormore of D94, T95, E115, N122, L125, E126, E127, E128, Q129, Q145, T154,T156, Q157, T159, D160, K163, T164, Y166, E196, or D197 (relative to SEQID NO: 1). In some embodiments, the mutation at one or more of D94, T95,E115, N122, L125, E126, E127, E128, Q129, Q145, T154, T156, Q157, T159,D160, K163, T164, Y166, E196, or D197 is not exclusively a mutation toalanine.

Alanine replacement mutations at F194; F190 and H191; K184, R186, andD187; R142 and R144 have been previously shown to decrease binding toF-pili, Deng & Perham, 2002. Thus, in some embodiments, a mutation ischosen from a mutation that does not include one or more of thefollowing residues: R142, R144, W181, K184, R186, D187, F190, H191, orF194 (numbering relative to SEQ ID NO: 1). However, replacement of R142,R144, W181, K184, R186, D187, F190, H191, or F194 with a non-alanineresidue may increase amyloid binding. Thus, in one embodiment, themutation is a non-alanine mutation at one or more of R142, R144, W181,K184, R186, D187, F190, H191, or F194. In one embodiment, the mutationis a non-alanine mutation at F194. In another embodiment, the mutationis a non-alanine mutation at F190 and H191. In another embodiment, themutation is a non-alanine mutation at K184, R186, and D187. In anotherembodiment, the mutation is a non-alanine mutation at W181. In anotherembodiment, the mutation is a non-alanine mutation at R142 and R144. Incertain embodiments, the mutation is not exclusively one, some, or allof: T13I, T101I, Q129H, G153D, W181A, F190A, F194A, and D209Y.

In some embodiments, the mutation is at one or more residues located onthe surface of the N2 domain, which is the portion of g3p that bindsF-pili. In one embodiment, the mutation is at one or more residueslocated on the outer rim of the N2 domain. In other embodiments, themutation is at one or more residues located on the surface of the N1domain, which is the portion of g3p that binds ToIA. In one embodiment,the mutation is at one or more residues located on the outer rim of theN1 domain. In another embodiment, the mutation is at one or more solventaccessible residues on g3p. In yet another embodiment, the mutation(s)shifts the cis/trans equilibrium at Pro213 to greater than 50, 60, 70,80, 90, or 95% trans. Thus, in some embodiments, the g3p is a mutatedg3p with a cis/trans equilibrium at Pro213 that is at least 50, at least60, at least 70, at least 80, at least 90, or at least 95% trans.

In some embodiments, the g3p mutant or amyloid binding fragment thereofdoes not include mutations at structurally conserved residues. Examplesof structurally conserved residues include residues that, despitepotential sequence insertions, are involved in providing domainstructure in both Ff and 1-family members.

In some embodiments, any mutation made preserves amyloid binding. Inother embodiments, the mutation does not replace a proline residue.

In some embodiments, any mutation made preserves amyloid binding anddoes not replace a cysteine residue. In some embodiments, the mutationpreserves all, at least one, at least two, at least three or all four ofthe disulphide bridges found within g3p. Thus, in one embodiment, anymutation preserves the two disulphide bridges in N1 between Cys7 andCys36 and between Cys46 and Cys53. In another embodiment, any mutationpreserves either, but not both, of the disulphide bridges in N1 betweenCys7 and Cys36 and between Cys46 and Cys53. In one embodiment, thedisulphide bridge between Cys188 and Cys201 is preserved. In someembodiments, each of the disulphide bridges Cys7 and Cys36, Cys46 andCys53, and Cys188 and Cys201 are preserved. In one embodiment, themutations preserve the disulphide bridge between Cys354 and Cys371. Insome embodiments, the mutations preserve the disulphide bridges betweenCys7 and Cys36, Cys46 and Cys53, Cys188 and Cys201, and Cys354 andCys371.

In some embodiments, any mutation made preserves amyloid binding anddecreases the melting temperature (T_(M)) of N1N2. T_(M) may be measuredusing any of the methods described in the Examples. Mutants thatdecrease the T_(M) of N1N2 are expected to exhibit better binding to Aβ,inhibit Aβ assembly to a greater extent, and to be at least as effectivein a disaggregation assay as g3p of M13. Accordingly, such mutants, aswell as fusion proteins and phage comprising these mutants are expectedto be at least as efficacious therapeutically as the correspondingsequences in M13, fusion proteins thereof, and intact M13, respectively,in treating one or more protein misfolding diseases.

Mutants may also be designed to include a targeting sequence. Suchtargeting sequences may be inserted into the flexible linker regionsbetween N1N2, or between N2 and another domain in an N2 fusion protein.Targeting nuclear localization sequences (NLS) might be beneficial inHuntington's disease. Targeting the endosome may be beneficial inParkinson's Disease's.

In addition to targeting specific regions in the cell, targetingsequences may be used to target different kinds of amyloid. Nucleatingsequences may increase affinity and direct the mutant protein to aparticular amyloid. Other mutants may be prepared that include peptidesequences that are so hydrophobic that they precipitate on their own.For example, multiple AVVAI sequences can be added to g3p and or amyloidbinding fragments thereof (e.g., N2 and N1N2) and/or their fusionproteins to generate chimeric proteins that have enhanced, multiplebinding sequences. Some examples of peptides that bind amyloid and maybe incorporated into the mutant or chimeric proteins comprising g3p, N2,and N1N2 and/or their fusion proteins are the peptide inhibitors basedon the GxFxGxF (SEQ ID NO: 21) framework described in Sato, Biochemistry(2006) 45:5503-16 and the KLVFF (SEQ ID NO: 22) peptide described inTjernberg et al., J. Biol. Chem. (1996) 271:8545-48. Other targetingmoieties are known and may also be used in the present invention. See,e.g., Sciarretta et al., Methods in Enzymology (2006) 413:273-312.

Amyloid-Binding Display Vehicles and Carriers

In another aspect of the invention g3p and amyloid-binding fragmentsthereof (including mutants and variants of any of the foregoing),including but not limited to N1N2 domains and N2 domains, as well asmolecules, polypeptides and fusion proteins that comprise them may becombined with other organic or even inorganic carriers that providemolecular scaffolds that preserve amyloid binding but provide additionalfeatures.

In some embodiments, the g3p or amyloid binding fragment thereof, or g3pfusion protein, and the carrier are covalently linked throughnon-recombinant means, such as, for example, a chemical linkage otherthan a peptide bond. Any suitable chemical crosslinker may be used. Anyknown methods of covalently linking polypeptides to other molecules (forexample, carriers) may also be used. In some embodiments, the g3p oramyloid binding fragment thereof, or g3p fusion protein, and the carriermay be fused through a linker that is comprised of at least one aminoacid or chemical moiety.

In some embodiments, the g3p or amyloid binding fragment thereof, or g3pfusion protein, and the carrier are noncovalently linked. In some suchembodiments, they may be linked, for example, using binding pairs.Exemplary binding pairs include, but are not limited to, biotin andavidin or streptavidin, an antibody and its antigen, etc.

Examples of carriers include, but are not limited to, viral particles(including phage, see below) in which a g3p protein or amyloid bindingfragment thereof not native to the virus is incorporated as part of theviral structure; polymers, whether natural, synthetic, or mixed;polymer-coated structures, such as beads (including surface derivatizedbeads); polyamino acids, nucleic acids; and liposomes. The carrier maybe linked either directly or indirectly to the g3p or amyloid-bindingfragment. Depending upon the carrier, intermediate linkages may be usedto provide appropriate spacing between the carrier and theamyloid-binding domain.

A polyaminoacid may be a carrier protein. Such polyaminoacids may bechosen from serum album (such as HSA), an additional antibody or portionthereof, for example the Fc region, fetuin A, fetuin B, leucine zippernuclear factor erythroid derivative-2 (NFE2), neuroretinal leucinezipper, tetranectin, or other polyaminoacids, for example, lysines. Thelocation of attachment of the polyaminoacid may be at the N terminus orC terminus, or other places in between, and also may be connected by achemical linker moiety to the g3p or amyloid binding fragment thereof.

In some embodiments, carriers include molecules with oligomerizationdomains. Oligomerization offers functional advantages when one of thefunctions of a protein or fragment thereof is binding, includingmultivalency, increased binding strength, and the combined function ofdifferent domains. These features are seen in natural proteins and mayalso be introduced by protein engineering. Accordingly, the inventionalso provides g3p and amyloid binding fragments (including mutants andvariants thereof) such as the N1N2 domain and N2 domain, comprising anoligomerization domain, for example, a dimerization domain. Suitableoligomerization domains include coiled-coil domains, includingalpha-helical coiled-coil domains; collagen domains; collagen-likedomains, and dimeric immunoglobulin domains. Suitable coiled-coilpolypeptide fusion partners of the invention include tetranectincoiled-coil domain, the coiled-coil domain of cartilage oligomericmatrix protein; angiopoietin coiled-coil domains; and leucine zipperdomains. When collagen or collagen-like oligomerization domains areused, they may comprise, for example, those found in collagens, mannosebinding lectin, lung surfactant proteins A and D, adiponectin, ficolin,conglutinin, macrophage scavenger receptor, and emilin. While some ofthese domains may be incorporated as fusion proteins, in manyembodiments they are non-recombinantly linked to the g3p, N1N2 domain,N2 domain or other amyloid-binding fragments, for example, throughcovalent bonding.

In addition, the invention provides g3p or amyloid-binding fragmentsthereof, or g3p fusion proteins, linked to a polymer. Polymers employedin the invention will be pharmaceutically acceptable for the preparationof a therapeutic product or composition.

Polymers are typically attached to a g3p or amyloid binding fragmentthereof with consideration of effects on functional or antigenic domainsof the polypeptide. In general, chemical derivatization may be performedunder any suitable condition used to react a protein with an activatedpolymer molecule. Activating groups which can be used to link thepolymer to the active moieties include sulfone, maleimide, sulfhydryl,thiol, triflate, tresylate, azidirine, oxirane, and 5-pyridyl.

Suitable, clinically acceptable, water soluble polymers include, but arenot limited to, polyethylene glycol (PEG), polyethylene glycolpropionaldehyde, copolymers of ethylene glycol/propylene glycol,monomethoxy-polyethylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, poly (β-aminoacids) (either homopolymers or random copolymers), poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers(PPG) and other polyakylene oxides, polypropylene oxide/ethylene oxidecopolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and otherpolyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylatedglucose, colonic acids or other carbohydrate polymers, Ficoll, ordextran and mixtures thereof.

PEG moieties of the invention may be branched or linear chain polymers.In an embodiment, the present invention contemplates a chemicallyderivatized polypeptide which includes mono- or poly- (e.g., 2-4) PEGmoieties. Pegylation may be carried out by any of the pegylationreactions known in the art. Methods for preparing a pegylated proteinproduct are generally known in the art. Optimal reaction conditions willbe determined on a case by case basis, depending on known parameters andthe desired result.

There are a number of PEG attachment methods available to those skilledin the art, for example, EP 0 401 384; Malik et al., Exp. Hematol.,(1992) 20:1028-1035; Francis, Focus on Growth Factors, 3:4-10 (1992); EP0 154 316; EP 0 401 384; WO 92/16221; WO 95/34326; and the otherpublications cited herein that relate to pegylation.

When a g3p, N1N2 domain, N2 domain or other amyloid-binding fragment (aswell as mutants and variants thereof and compounds, polypeptides andfusion proteins comprising any of the foregoing) is PEGylated, the PEGmay be attached by either chemically derivatizing the g3p, N1N2 domain,N2 domain or other amyloid-binding fragment. In other embodiments, anamino acid residue suitable for modification by a PEG molecule may berecombinantly introduced into the g3p, N1N2 domain, N2 domain or otheramyloid-binding fragment.

Pegylation may be performed via an acylation reaction or an alkylationreaction with a reactive polyethylene glycol molecule. Thus, proteinproducts of the present invention include pegylated proteins wherein thePEG groups are attached via acyl or alkyl groups. Such products may bemono-pegylated or poly-pegylated (for example, those containing 2-6 or2-5 PEG groups). An example of a suitable activated PEG ester is PEGesterified to N-hydroxysuccinimide (NHS).

Pegylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with a polypeptide in the presence of a reducingagent. For the reductive alkylation reaction, the polymer(s) selectedshould have a single reactive aldehyde group. An exemplary reactive PEGaldehyde is polyethylene glycol propionaldehyde, which is water stable,or mono C₁-C₁₀ alkoxy or aryloxy derivatives thereof, see for example,U.S. Pat. No. 5,252,714.

In some embodiments, g3p, N1N2 domain, N2 domain or otheramyloid-binding fragment is expressed as part of a phage and the g3p,N1N2 domain, N2 domain or other amyloid-binding fragment is prepared byisolating it from the phage particles. In general, however, recombinanttechniques are used to prepare the g3p, amyloid binding fragment of g3p(including mutants and variants thereof). In general, the resultingprotein is isolated prior to combining with a carrier.

In some embodiments, the display vehicle is a phage. In theseembodiments, a gene encoding a g3p protein, N1N2 domain, N2 domain, andother amyloid-binding fragment (including mutants and variants of allthe foregoing) is incorporated into a bacteriophage genome and expressedas part of the phage. For example, in one embodiment, a mutant g3pprotein with higher amyloid-binding affinity than the g3p of M13 phageis used to replace the wild type g3p of M13 phage. The resulting phagethus also has improved binding relative to wild type M13. However, anyof the g3p or amyloid binding fragments described may be incorporatedinto a phage. In these embodiments, the wild type gene 3 may be replacedentirely by a g3p of the invention. Alternatively, as discussed forphage with increased copy number of g3p, the recombinant molecule may befused to a gene encoding a phage coat protein (including wild type g3p)and displayed on the phage in a manner analogous to antigen and antibodychains in phage display libraries. Any filamentous bacteriophage may bemodified to express a g3p of the invention, including, but not limitedto M13, fd, f1, I22, Ike, or If1. In some embodiments, a helper phagemay be used in conjunction with the modified phage.

Recombinant Techniques

In general a DNA encoding a g3p protein or amyloid binding fragmentthereof (as well as mutants and variants thereof and compounds,polypeptides and fusion proteins comprising any of the foregoing) isprepared using conventional recombinant DNA techniques, such as cloningof the g3p gene, direct DNA synthesis, or by isolating the correspondingDNA from a library using, for example, the M13 sequence as a probe.(See, e.g., Sambrook et al. 1989, Molecular Cloning A Laboratory Manual,Cold Spring Harbor Laboratory, N.Y. and Ausubel et al. 1989, CurrentProtocols in Molecular Biology, Greene Publishing Associates and WileyInterscience, N.Y.).

For recombinant production, a nucleic acid sequence encoding a g3p oramyloid binding fragment thereof is inserted into an appropriateexpression vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence, or in thecase of an RNA viral vector, the necessary elements for replication andtranslation. The encoding nucleic acid is inserted into the vector inproper reading frame.

Accordingly, the invention provides vectors comprising polynucleotidesthat encode g3p or an amyloid binding fragment thereof (includingmutants and variants thereof). Vectors comprising polynucleotides thatencode a g3p or g3p-fusion molecule are also provided. Such vectorsinclude, but are not limited to, DNA vectors, phage vectors, viralvectors, retroviral vectors, etc.

In some embodiments, a vector is selected that is optimized forexpression of polypeptides in CHO or CHO-derived cells. Exemplary suchvectors are described, e.g., in Running Deer et al., Biotechnol. Prog.(2004) 20:880-889.

In some embodiments, a vector is chosen for in vivo expression of g3p,amyloid binding fragment thereof and/or g3p fusion molecules in animals,including humans. In some such embodiments, expression of thepolypeptide is under the control of a promoter that functions in atissue-specific manner.

Expression vectors are transfected or co-transfected into a suitabletarget cell, which will express the polypeptides. Nonlimiting exemplarytransfection methods are described, e.g., in Sambrook et al., MolecularCloning, A Laboratory Manual, 3^(rd) ed. Cold Spring Harbor LaboratoryPress (2001). Nucleic acids may be transiently or stably transfected inthe desired host cells, according to methods known in the art. A varietyof host-expression vector systems may be utilized to express theproteins described herein including either prokaryotic or eukaryoticcells. These include, but are not limited to, microorganisms such asbacteria (e.g., E. coli) transformed with recombinant bacteriophage DNAor plasmid DNA expression vectors containing an appropriate codingsequence; yeast or filamentous fungi transformed with recombinant yeastor fungi expression vectors containing an appropriate coding sequence;insect cell systems infected with recombinant virus expression vectors(e.g., baculovirus) containing an appropriate coding sequence; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus or tobacco mosaic virus) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containing anappropriate coding sequence; or animal cell systems, including mammaliancells (e.g., CHO, Cos, HeLa cells). The proteins may also be producedrecombinantly in duckweed. See, e.g., U.S. Pat. No. 8,022,270.

Vectors used in transformation will usually contain a selectable markerused to identify transformants. In bacterial systems, this can includean antibiotic resistance gene such as ampicillin or kanamycin.Selectable markers for use in cultured mammalian cells include genesthat confer resistance to drugs, such as neomycin, hygromycin, andmethotrexate. The selectable marker may be an amplifiable selectablemarker. One amplifiable selectable marker is the DHFR gene. Anotheramplifiable marker is the DHFRr cDNA (Simonsen and Levinson, Proc. Natl.Acad. Sci. (USA), (1983) 80:2495). Selectable markers are reviewed byThilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham,Mass.) and the choice of selectable markers is well within the level ofordinary skill in the art.

The expression elements of the expression systems vary in their strengthand specificities. Depending on the host/vector system utilized, any ofa number of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedron promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g., heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5 K promoter) may beused; when generating cell lines that contain multiple copies ofexpression product, SV40-, BPV- and EBV-based vectors may be used withan appropriate selectable marker.

In cases where plant expression vectors are used, the expression ofsequences encoding linear or non-cyclized forms of the expressionproduct of the invention may be driven by any of a number of promoters.For example, viral promoters such as the 35S RNA and 19S RNA promotersof CaMV (Brisson et al., Nature (1984) 310:511-514), or the coat proteinpromoter of TMV (Takamatsu et al., EMBO J. (1987) 6:307-311) may beused; alternatively, plant promoters such as the small subunit ofRUBISCO (Coruzzi et al., EMBO J. (1984) 3:1671-1680; Broglie et al.,Science (1984) 224:838-843) or heat shock promoters, e.g., soybeanhsp17.5-E or hsp17.3-B (Gurley et al., Mol. Cell. Biol. (1986)6:559-565) may be used. These constructs can be introduced into plantcells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNAtransformation, microinjection, electroporation, etc. For reviews ofsuch techniques see, e.g., Weissbach & Weissbach 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp. 421-463; andGrierson & Corey 1988, Plant Molecular Biology, 2d Ed., Blackie, London,Ch. 7-9.

In one insect expression system that may be used to produce proteins ofthe invention, Autographa californica nuclear polyhidrosis virus (AcNPV)is used as a vector to express the foreign genes. The virus grows inSpodoptera frugiperda cells. A coding sequence may be cloned intonon-essential regions (for example, the polyhedron gene) of the virusand placed under control of an AcNPV promoter (for example, thepolyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e. virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (see, e.g., Smith et al., J. Virol. (1983) 46:584;U.S. Pat. No. 4,215,051). Further examples of this expression system maybe found in Ausubel et al., eds. 1989, Current Protocols in MolecularBiology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This fusion gene may then be inserted in theadenovirus genome by in vitro or in vivo recombination. Insertion in anon-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts (see, e.g., Logan & Shenk, Proc. Natl. Acad.Sci. (USA) (1984) 81:3655). Alternatively, the vaccinia 7.5 K promotermay be used (see, e.g., Mackett et al., Proc. Natl. Acad. Sci. (USA)(1982) 79:7415; Mackett et al., J. Virol. (1984) 49:857; Panicali etal., Proc. Natl. Acad. Sci. (USA) (1982) 79:4927). Other viralexpression systems include adeno-associated virus and lentiviruses.

Host cells containing the DNA constructs are grown in an appropriategrowth medium. As used herein, the term “appropriate growth medium”means a medium containing nutrients required for the growth of cells.The recombinantly produced protein of the invention can be isolated fromthe culture media using techniques conventional in the art.

In Vitro Assays

In some embodiments, disaggregation of amyloid may be monitored usingthe Thioflavin T Fluorescence (ThT) assay.

In some embodiments, disaggregation is tested by monitoring detergentsolubilization in the presence or absence of a composition of theinvention. For example, aggregated α-synuclein can be treated with acomposition of the invention. A composition that disaggregates theaggregated α-synuclein will cause the α-synuclein fibers to solubilizefaster in detergents such as SDS, compared to untreated fibers. Thisconversion of the amyloid fibers into soluble forms can be monitored byincorporating a proportion of labeled (e.g., with Cy5) α-synucleinmonomers during aggregation.

In some embodiments, preventing the formation of toxic amyloid oligomersis tested by a neuronal cell culture cytotoxicity assay. In this assay,differentiated N2a neuroblastoma cells or equivalents are coincubatedwith Aβ42 oligomers. The oligomers bind membranes and cause membraneperturbation and the leaking of cytosolic enzymes into the media.Prolonged incubation with high concentrations of oligomers will killcells. When oligomers are pre-treated with phage or g3p prior toincubating with cells, the oligomers are at least less toxic andsometimes nontoxic. This neutralizing effect may be quantitated bymeasuring the release of adenylate kinase, one exemplary cytosolicenzyme released by the neuronal cells after membrane perturbation.

In some embodiments, a composition of the invention inhibits conversionof soluble prion protein into proteinase K resistant conformer in theprotein misfolding cyclic amplification (PMCA) assay. Wang et al.,Science, (2010) 327:1132-35. In this assay, recombinant PrP is mixedwith the lipid POPG and RNA in either the presence or absence of acomposition of the invention. The material is then subjected to multiple(e.g., 48) cycles of a 30 second sonication followed by 29.5 minuteincubation. A fraction of the reaction mixture is then used to seedanother substrate tube and the cycle repeated. Each round is tested forthe presence of proteinase K resistant material, which is indicative ofthe infectious form of PrP. Reduction in proteinase K resistant materialin the presence of a composition of the invention indicates that thecomposition inhibits formation of the PK resistant conformer.

As noted above, amyloid forms of certain prion proteins, such as yeastprion protein NM, can also be detected in the filter trap assay.Accordingly, depending upon the prior protein, in some embodiments theability of a composition of the invention to disaggregate prion proteinaggregates may be tested in the filter trap assay.

In Vivo Functional Assays

In addition to activities such as increased binding affinity for amyloidor decrease in T_(M), that can be demonstrated in in vitro assays,compositions of the invention may also reduce amyloid in one of severalin vivo assays. One method for determining amyloid reduction in vivouses positron emission tomography (PET) with the imaging agentflorbetapir (F18-AV-45, Eli Lilly) before and after treatment to comparethe number and/or distribution of β-amyloid. Of course, as additionalbiomarkers are identified, they may also be used to measure reduction ofamyloid.

Another method of determining whether a composition reduces amyloid invivo uses the hAPP mouse model. Rockenstein, J Neurosci Res. (2001)66(4):573-82. These mice develop high levels of β-amyloid at an earlyage (3-4 months). The ability of a composition to reduce amyloid can bedetermined by injecting mice with a composition of the invention thencomparing levels of amyloid in those mice compared to non-injectedcontrols. It is also possible to inject a composition into only onehemisphere of an hAPP mouse, allowing comparison of amyloid levelsbetween injected and non-injected hemispheres in the same mouse.

In another example, compositions of the invention are tested in thetransgenic mouse model for Alzheimer's disease (TgAD) described inUS2011/0142803, Hsiao et al., Science (1996) 274:99-102, or Duyckaertset al., Acta Neuropathol (2008) 115:5-38. Briefly, wild-type, as well astransgenic mice, are challenged. To assess the potential of acomposition of the invention to act as disaggregating agent, acomposition is injected intracranially to transgenic mice (Taconic,APPSWE(2576), 10 month-old). For example, for compositions comprisingphage, 2.5 μl the filamentous phage solution (10¹⁴ phages/ml) areinjected over 10 minutes (Bregma −2.8 mm, lateral 2.5 mm, ventral 2.5mm) to one hemisphere, while to the contra-lateral side,phosphate-buffered-saline (PBS) is applied as a control. Treated miceare then sacrificed at different time points and brains post-fixedovernight in 4% paraformaldehyde, and cut using a microtome.Thioflavin-S(ThS) staining is performed to evaluate amyloid load.Sections are stained with Mayer's hematoxylin to quench nuclearautofluorescence and after washing ThS solution (1%) is applied for 3minutes. Differentiation is done using 1% acetic acid for 20 min, andafter washes the slides are dried and mounted with anti fade mountingmedium. Amyloid load is calculated using LEICA Qwin program.Alternatively, amyloid load can be assessed with an anti-amyloidantibody.

Biodistribution of radioactive (e.g., I¹²⁵) or fluorescently labeledcompositions, or unlabelled compositions, including filamentous phage,can also be measured to show that a composition binds amyloid in vivo.For example, when the composition comprises phage, the filamentous phagemay be radioactively or fluorescently labeled. BALB/c mice are dividedinto groups. Each mouse then receives intranasally 100 μl of phage(1.25×10¹² phage) over an hour. The first group of mice is sacrificed anhour after administration of intra-cardial perfusion using 4%paraformaldehyde. The second group is sacrificed 3 hours post-treatment,and the last group, after 24 hours. After perfusion, brains as well asperipheral organs are removed and the label is measured. Alternatively,unlabelled compositions or phage can be assessed for binding usingsimilar methods but co-staining brain sections with a stain thatrecognizes amyloid and a stain that recognizes the composition or phage.

Intranasal administration of filamentous phage is also used to fullyevaluate compositions comprising phage, such as phage comprising amutant g3p or amyloid-binding fragment of g3p, or phage with anincreased number of g3p relative to wild type phage, as provided by theinvention. For example, phage are administered intranasally toSWE/APP2576 transgenic mice (Taconic, 10 month-old), a mouse model ofAlzheimer's Disease. Twenty microliters of phage solution (5×10¹²/ml)are applied every two weeks, for 4 to 12 months and cognitive functionsare evaluated. After the treatment period, a novel object recognitiontest is carried out to study the influence of phage treatment on memoryimprovement. On the first day, mice are exposed to two new objects for20 minutes. On the following day, one object is replaced, and thecuriosity of the mice to explore the novel item is tested. A recognitionindex is calculated for each mouse by dividing the time it spent nearthe new object by the total time spent near both objects. Thus, valuesabove 0.5 are indicative for recognizing the old item and spending moretime around the new object for its investigation.

Other transgenic models of protein misfolding disease may also be usedto demonstrate that a composition of the invention reduces amyloid. Nonlimiting examples include the “D line” α-synuclein mice (a model ofParkinson's Disease, Masliah et al., Science (2000) 287:1265-1269);Tg2576 mice (a model of Alzheimer's Disease, Hsiao et al., Science(1996) 274:99-102 and Duyckaerts et al., Acta Neuropathol (2008)115:5-38 at 9); various Jax® Mice for Parkinson's Disease Research(Jackson Laboratories, Bar Harbor, Me.); and mouse and rat modelsavailable from JSW Lifescience, including those for Parkinson's Disease,Alzheimer's Disease, Huntington's Disease.

Phage in which g3p has been rendered inactive are expected to beinactive in these assays, whereas wild type phage co-localize toamyloid, reduce amyloid load, prevent amyloid formation, and/or removetoxic oligomers and result in improvement in cognitive function. Phagecomprising a g3p or an amyloid-binding fragment thereof, as provided bythe invention, can thus be tested for in vivo activity relative to thesenegative and positive controls.

Pharmaceutical Compositions

In another aspect, the invention provides pharmaceutically acceptablecompositions comprising any of the above-described agents of theinvention (i.e., (a) g3p, amyloid binding fragments of g3p, or mutantsor variants thereof; (b) compounds, polypeptides and fusion proteinscomprising g3p, amyloid binding fragments of g3p, or mutants or variantsthereof; (c) filamentous bacteriophage bearing an increased number ofcopies of g3p as compared to wild-type phage; (d) amyloid bindingdisplay vehicles bearing g3p, amyloid binding fragments of g3p, mutantsor variants thereof, or compounds, polypeptides and fusion proteinscomprising g3p, amyloid binding fragments of g3p, or mutants or variantsthereof; or (e) modified filamentous phage bearing variants of g3p,amyloid binding fragments of g3p (not as part of a displayed g3pprotein), mutants or variants of such binding fragments, or fusionproteins or other heterologous polypeptides that comprise g3p, amyloidbinding fragments of g3p, or mutants or variants thereof).

A “pharmaceutical composition” refers to a therapeutically effectiveamount of a composition as described herein with a physiologicallysuitable carrier and/or excipient. A pharmaceutical composition does notcause significant irritation to an organism. The phrases“physiologically acceptable carrier” and “pharmaceutically acceptablecarrier” which may be used interchangeably refer to a carrier or adiluent that does not cause significant irritation to an organism anddoes not abrogate the biological activity and properties of theadministered composition.

The term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, include, for example,saline, calcium carbonate, calcium phosphate, various sugars and typesof starch, cellulose derivatives, gelatin, vegetable oils, polyethyleneglycols, and surfactants, including, for example, polysorbate 20.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in a conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intocompositions which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen and upon the nature ofthe composition delivered (e.g., protein versus phage).

Suitable routes of administration for the pharmaceutical compositions ofthe invention may, for example, include transmucosal, especiallytransnasal delivery; parenteral delivery, including intramuscular,subcutaneous, intramedullary, intrathecal, intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections;oral; or rectal delivery.

In some embodiments, a pharmaceutical composition is administered in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into the brain of a patient. In someembodiments, the injection technique is any technique that avoids theblood-brain barrier, for example, by direct intramedullary, intrathecal,or intraventricular injection.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

In some embodiments, a pharmaceutical composition of the invention isadministered via intranasal administration. Intranasal delivery has beenreported to enable the direct entry of viruses and macromolecules intothe cerebrospinal fluid (CSF) or CNS. Mathison et al, 1998; Chou et al,1997; Draghia et al, 1995.

For administration by the intranasal route, compositions areconveniently delivered in the form of an aerosol spray from apressurized pack or a nebulizer with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The various proteins described herein as components of pharmaceuticalcompositions may also be delivered to the brain using olfactory receptorneurons as a point of delivery. For example, an adenovirus vectorcomprising a gene encoding any of those proteins may be delivered viaolfactory receptor neurons. Draghia et al, 1995.

The compositions described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Pharmaceutical compositions for parenteral administration includeaqueous solutions of the composition in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asoily or water based injection suspensions. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacids esters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol or dextran. Optionally, the suspension may also containsuitable stabilizers or agents (e.g., surfactants such as polysorbate(Tween 20)) which increase the solubility of the active ingredients toallow for the preparation of highly concentrated solutions. A proteinbased agent such as, for example, albumin may be used to preventadsorption of M13 to the delivery surface (i.e., IV bag, catheter,needle, etc.).

For oral administration, the compositions can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art.

Formulations may be presented in unit dosage form, e.g., in vials,ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents. Singledosage forms may be in a liquid or a solid form. Single dosage forms maybe administered directly to a patient without modification or may bediluted or reconstituted prior to administration. In certainembodiments, a single dosage form may be administered in bolus form,e.g., single injection, single oral dose, including an oral dose thatcomprises multiple tablets, capsule, pills, etc. In alternateembodiments, a single dosage form may be administered over a period oftime, such as by infusion, or via an implanted pump, such as an ICVpump. In the latter embodiment, the single dosage form may be aninfusion bag or pump reservoir pre-filled with the indicated number offilamentous bacteriophage. Alternatively, the infusion bag or pumpreservoir may be prepared just prior to administration to a patient bymixing a single dose of the filamentous bacteriophage with the infusionbag or pump reservoir solution.

Another aspect of the invention includes methods for preparing apharmaceutical composition of the invention. Techniques for formulationof drugs may be found, for example, in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition, which isincorporated herein by reference in its entirety.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.

Determination of a therapeutically or diagnostically effective amount iswell within the capability of those skilled in the art, especially inlight of the detailed disclosure provided herein.

Dosage amount and interval may be adjusted individually to provide brainlevels of the phage display vehicle which are sufficient to treat ordiagnose a particular brain disease, disorder, or condition (minimaleffective concentration, MEC). The MEC will vary for each preparation,but can be estimated from in vitro data. Dosages necessary to achievethe MEC will depend on individual characteristics.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, which maintainsbrain levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated or diagnosed, the severity of theaffliction, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

It is to be understood that both the foregoing and following descriptionare exemplary and explanatory only and are not restrictive of theinvention, as claimed.

Therapeutic Uses

As noted, filamentous bacteriophage M13, and related filamentous phage,have shown utility in animal models of protein misfolding disease. SeeUnited States patent publication US 2011/0142803, incorporated byreference herein in its entirety. In particular, it has been discoveredthat filamentous bacteriophage have the ability to disaggregate amyloidthat have already formed in the brain. Removal of amyloid is expected toreduce, slow the progression of, or even to reverse the symptomsassociated with a variety of diseases characterized by misfolded and/oraggregated proteins in the brain. See, e.g., WO2006083795 andWO2010060073, incorporated by reference herein in their entirety.

Further, M13 has been shown to disaggregate at least four differentamyloid fibers: amyloid-β 1-42 fibers (fAβ42), α-synuclein fibers(fαsyn), yeast prion NM fibers (fNM), and tau fibers (ftau).

Accordingly, another aspect of the invention relates to the use of anyof the compositions of the invention, such as those comprising g3p, N1N2domain, N2 domain or other amyloid-binding fragments (including mutantsor variants of all of the foregoing), or g3p fusion proteins, displayvehicles, or phage comprising any of the above, in the treatment ofprotein misfolding diseases, including, but not limited to, thosediseases involving any of fAβ42, fαsyn, fNM, or ftau.

In the context of treatments, the terms “patient”, “subject” and“recipient” are used interchangeably and include humans as well as othermammals. In some embodiments, a patient is a human who is positive for abiomarker associated with a protein misfolding disease. In oneembodiment, the patient exhibits β-amyloid deposits as detected by PETimaging with florbetapir.

The term “treating” is intended to mean reducing, slowing, or reversingthe progression of a disease in a patient exhibiting one or moreclinical symptoms of a disease. “Treating” is also intended to meanreducing, slowing, or reversing the symptoms of a disease in a patientexhibiting one more more clinical symptoms of a disease. In oneembodiment, the patient exhibits β-amyloid deposits as detected by PETimaging with florbetapir and the number of β-amyloid deposits is reducedby the treatment. In one embodiment, the patient exhibits β-amyloiddeposits as detected by the g3p compositions of the present inventionand the number of β-amyloid deposits are reduced or maintained by thetreatment. In another embodiment, the patient exhibits any type ofamyloid deposits as detected by PET imaging and the cognitive functionof the patient is improved by the treatment. Improvement in cognitivefunction may be assayed by the methods and tests of McKhann et al.,Alzheimer's & Dementia (2011) May; 7(3):263-9.

“Prophylaxis” is distinct from treating and refers to administration ofa composition to an individual before the onset of any clinicalsymptoms. Prophylaxis using any of the g3p and/or ToIA compositions ofthe present invention is encompassed. Prophylaxis may be implicated inindividuals who are known to be at increased risk for a disease, or whomare certain to develop a disease, solely on the basis of one or moregenetic markers. Many genetic markers have been identified for thevarious protein misfolding diseases. For examples, individuals with oneor more of the Swedish mutation, the Indiana mutation, or the Londonmutation in human amyloid precursor protein (hAPP) are at increased riskfor developing early-onset Alzheimer's Disease and so are candidates forprophylaxis. Likewise, individuals with the trinucleotide CAG repeat inthe huntingtin gene, particularly those with 36 or more repeats, willeventually develop Huntington's Disease and so are candidates forprophylaxis.

In some embodiments, a protein or fragment is used directly as atherapeutic. In these embodiments, a g3p, N1N2 domain, N2 domain, orother amyloid-binding fragments (including mutants or variants of any ofthe foregoing) is directly incorporated into a pharmaceuticalcomposition or formulation. In other embodiments, a g3p, N1N2 domain, N2domain, or other amyloid-binding fragment (including mutants or variantsof any of the foregoing) is part of a fusion protein or display vehicle,such as a phage, and in these embodiments it is the fusion protein ordisplay vehicle that is incorporated into a pharmaceutical compositionor formulation of the invention. In other embodiments, the compositioncomprises a phage comprising g3p or amyloid binding fragment thereofthat is more efficacious than g3p of wild type M13 phage in reducing ormaintaining levels of amyloid. In some embodiments, the phage that ismore efficacious in reducing or maintaining levels of amyloid than M13expresses more than 5 copies of a g3p.

The term “protein misfolding” refers to diseases characterized byformation of amyloid protein by an aggregating protein (amyloid formingpeptide), such as, but not limited to, β-amyloid, serum amyloid A,cystatin C, IgG kappa light chain, or a prion protein. Diseases known tobe associated with misfolded and/or aggregated amyloid protein includeAlzheimer's disease, which includes early onset Alzheimer's disease,late onset Alzheimer's disease, and presymptomatic Alzheimer's disease,Parkinson's disease, SAA amyloidosis, cystatin C, hereditary Icelandicsyndrome, senility, multiple myeloma, prion diseases including but notlimited to kuru, Creutzfeldt-Jakob disease (CJD),Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia(FFI), scrapie, and bovine spongiform encephalitis (BSE); amyotrophiclateral sclerosis (ALS), spinocerebellar ataxia (SCA1), (SCA3), (SCA6),(SCA7), Huntington disease, entatorubral-pallidoluysian atrophy, spinaland bulbar muscular atrophy, hereditary cerebral amyloid angiopathy,familial amyloidosis, frontotemporal lobe dementia, British/Danishdementia, and familial encephalopathy. The g3p compositions of theinvention may be used to treat “protein misfolding” diseases.

Many of these misfolded and/or aggregated amyloid protein diseases occurin the central nervous system (CNS). Some examples of diseases occurringin the CNS are Parkinson's Disease; Alzheimer's Disease; frontotemporaldementia (FTD) including those patients having the following clinicalsyndromes: behavioral variant FTD (bvFTD), progressive non-fluentaphasia (PNFA) and semantic dementia (SD); frontotemporal lobardegenerations (FTLDs); and Huntington's Disease. The g3p compositions ofthe invention may be used to treat diseases characterized by misfoldedand/or aggregated amyloid protein that occur in the central nervoussystem (CNS).

Misfolding and/or aggregation of proteins may also occur outside theCNS. Amyloidosis A (AA) (for which the precursor protein is serum acutephase apolipoprotein, SAA) and multiple myeloma (precursor proteinsimmunoglobulin light and/or heavy chain) are two widely known proteinmisfolding and/or aggregated protein diseases that occur outside theCNS. Other examples include disease involving amyloid formed byβ₂-microglobulin, transthyretin (Familial Amyloidotic Polyneuropathy[FAP], Familial Amyloidotic Cardiomyopathy [FAC], and Senile SystemicAmyloidosis [SSA]), (apo)serum AA, apolipoproteins Al, All, and AIV,gelsolin (Finnish form of Familial Amyloidotic Polyneuropathy),lysozyme, firbrinogen, cystatin C (Cerebral Amyloid Angiopathy,Hereditary Cerebral Hemorrhage with Amyloidosis, Icelandic Type),(pro)calcitonin, islet amyloid polypeptide (IAPP amyloidosis), atrialnatriuretic factor, prolactin, insulin, lactadherin, kerato-epithelin,lactoferrin, odontogenic ameloblast-associated protein, and semenogelinI. The g3p compositions of the invention may be used to treat diseasesinvolving misfolding and/or aggregation of proteins that occur outsidethe CNS.

Neurodegenerative diseases may also involve tau lesions. (Reviewed inLee et al. (2001) Annu. Rev. Neurosci. 24:1121-159). Tau proteins aremicrotubule-associated proteins expressed in axons of both central andperipheral nervous system neurons. Neurodegenerative tauopathies(sometimes referred to as tauopathies) are encompassed. Examples oftauopathies include Alzheimer's Disease, Amyotrophic lateralsclerosis/parkinsonism-dementia complex, Argyrophilic grain dementia,Corticobasal degeneration, Creutzfeldt-Jakob disease, Dementiapugilistica, diffuse neurofibrillary tangles with calcification, Down'ssyndrome, Frontotemporal dementias including frontotemporal dementiawith parkinsonism linked to chromosome 17,Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease,Myotonic dystrophy, Niemann-Pick disease type C, Non-Guamanian motorneuron disease with neurofibrillary tangles, Pick's disease,Postencephalitic parkinsonism, Prion protein cerebral amyloidangiopathy, Progressive subcortical gliosis, Progressive supranuclearpalsy, Subacute sclerosing panencephalitis, and Tangle only dementia.Some of these diseases may also include deposits of fibrillar amyloid βpeptides. For example, Alzheimer's disease exhibits both amyloid βdeposits and tau lesions. Similarly, prion-mediated diseases such asCreutzfeldt-Jakob disease, prion protein cerebral amyloid angiopathy,and Gerstmann-Sträussler-Scheinker syndrome may have also have taulesions. Thus an indication that a disease is a “tauopathy” should notbe interpreted as excluding the disease from other neurodegenerativedisease classifications or groupings, which are provided merely as aconvenience. The g3p compositions of the invention may be used to treatneurodegenerative diseases as well as diseases involving tau lesions.

In one embodiment, a pharmaceutical composition or formulation is foruse in a method of reducing amyloid in a patient exhibiting symptomsrelated to the presence of amyloid or that is positive for a biomarkerassociated with a protein misfolding disease, such as florbetapir(AV-45, Eli Lilly), comprising administering to the patient an effectiveamount of a pharmaceutical composition or formulation as describedherein. In one embodiment, the route of administration is selected fromintrathecal injection, direct intraventricular injection,intraparenchymal injection, or intranasal delivery.

In one embodiment, a pharmaceutical composition or formulation is foruse in a method of maintaining the level of amyloid in a patientexhibiting symptoms related to the presence of amyloid or that ispositive for a biomarker associated with a protein misfolding disease,such as florbetapir (AV-45, Eli Lilly), comprising administering to thepatient an effective amount of a pharmaceutical composition orformulation as described herein. In one embodiment, the route ofadministration is selected from intrathecal injection, directintraventricular injection, intraparenchymal injection, or intranasaldelivery.

In one embodiment, a pharmaceutical composition or formulation is foruse in a method of disaggregating amyloid in a patient comprisingadministering to a patient having amyloid an effective amount of apharmaceutical composition or formulation as described herein. In oneembodiment, the route of administration is selected from intrathecalinjection, direct intraventricular injection, intraparenchymalinjection, or intranasal delivery.

In one embodiment, a pharmaceutical composition or formulation is foruse in a method of causing the disaggregation of β-amyloid deposits inthe brain, comprising injecting directly into the brain of a patient inneed thereof an effective amount of pharmaceutical composition asdescribed herein, thereby causing a reduction in β-amyloid deposits inthe brain.

In one embodiment, a pharmaceutical composition or formulation is foruse in a method of reducing amyloid formation in the brain. Reducingamyloid formation in the brain may prevent, treat or reduce the symptomsor severity of a protein-misfolding or neurodegenerative disease. In oneembodiment, the route of administration is selected from intrathecalinjection, direct intraventricular injection, intraparenchymalinjection, or intranasal delivery.

In one embodiment, a pharmaceutical composition or formulation of theinvention is for use in a method for promoting amyloid clearance in thebrain. Promoting amyloid clearance may prevent, treat or reduce thesymptoms or severity of a protein-misfolding or neurodegenerativedisease. In one embodiment, the route of administration is selected fromintrathecal injection, direct intraventricular injection,intraparenchymal injection, or intranasal delivery.

In one embodiment, a pharmaceutical composition or formulation of theinvention is for use in a method for inhibiting amyloid aggregation inthe brain. Inhibiting amyloid aggregation in the brain may prevent,treat or reduce the symptoms or severity of a protein-misfolding orneurodegenerative disease. In one embodiment, the route ofadministration is selected from intrathecal injection, directintraventricular injection, intraparenchymal injection, or intranasaldelivery.

In one embodiment, a pharmaceutical composition or formulation of theinvention is for use in a method for clearing toxic amyloid oligomers inthe brain. Clearing toxic amyloid oligomers in the brain may prevent,treat or reduce the symptoms or severity of a protein-misfolding orneurodegenerative disease. In one embodiment, the route ofadministration is selected from intrathecal injection, directintraventricular injection, intraparenchymal injection, or intranasaldelivery.

In one embodiment, a pharmaceutical composition or formulation of theinvention is for use in a method for preventing the formation of toxicamyloid oligomers in the brain. Preventing the formation of toxicoligomers in the brain may prevent, treat or reduce the symptoms orseverity of a protein-misfolding or neurodegenerative disease. In oneembodiment, the route of administration is selected from intrathecalinjection, direct intraventricular injection, intraparenchymalinjection, or intranasal delivery.

In one embodiment, a pharmaceutical composition or formulation of theinvention is for use in a method for protecting neurons from amyloiddamage. Protecting neurons from amyloid damage may prevent, treat orreduce the symptoms or severity of a protein-misfolding orneurodegenerative disease. In one embodiment, the route ofadministration is selected from intrathecal injection, directintraventricular injection, intraparenchymal injection, or intranasaldelivery. In one embodiment, a pharmaceutical composition or formulationof the invention for use in protecting neurons from amyloid damage isgiven prophylactically.

In some embodiments, the patient is positive for a biomarker associatedwith a protein misfolding and/or aggregation disease. In one embodiment,the biomarker is florbetapir (AV45, Eli Lilly).

In some embodiments, the patient is exhibiting symptoms of aneurodegenerative disease that is associated with the presence ofamyloid. In various embodiments, the amyloid is any of fAβ42, fαsyn,fNM, or ftau.

In certain embodiments, the neurodegenerative disease is Parkinson'sdisease, Alzheimer's disease, or Huntington's disease. In oneembodiment, the neurodegenerative disease is Alzheimer's disease. In oneembodiment, the neurodegenerative disease is Alzheimer's disease and thepatient exhibits β-amyloid as detected by the imaging agent florbetapir(AV-45, Eli Lilly).

In some embodiments, the patient is exhibiting symptoms of aprion-mediated disease.

In certain embodiments, the prion-mediated disease is chosen fromCreutzfeldt-Jakob disease, kuru, fatal familial insomnia, orGerstmann-Sträussler-Scheinker syndrome.

In some embodiments, the patient is exhibiting symptoms of aneurodegenerative tauopathy other than Alzheimer's disease. In certainembodiments, the disease to be treated is selected from Argyrophilicgrain dementia, Corticobasal degeneration, Dementia pugilistica, diffuseneurofibrillary tangles with calcification, Down's syndrome,Frontotemporal dementias including frontotemporal dementia withparkinsonism linked to chromosome 17, Hallervorden-Spatz disease,Myotonic dystrophy, Niemann-Pick disease type C, Non-Guamanian motorneuron disease with neurofibrillary tangles, Pick's disease,Postencephalitic parkinsonism, Progressive subcortical gliosis,Progressive supranuclear palsy, Subacute sclerosing panencephalitis, andTangle only dementia.

Diagnostics

In another aspect of the invention, the g3p and ToIA compositionsdescribed herein, including g3p fusion proteins, are used in diagnosticapplications associated with the various diseases described herein. Forexample, binding of a composition of the invention when used as animaging agent either in vivo or in vitro may be part of a diagnosis ofone of the protein misfolding diseases described.

Diagnostic agents, otherwise referred to herein as diagnosticcompositions, are encompassed, and may comprise any of theabove-described agents of the invention (i.e., (a) g3p, amyloid bindingfragments of g3p, or mutants or variants thereof; (b) compounds,polypeptides and fusion proteins comprising g3p, amyloid bindingfragments of g3p, or mutants or variants thereof; (c) filamentousbacteriophage bearing an increased number of copies of g3p as comparedto wild-type phage; (d) amyloid binding display vehicles bearing g3p,amyloid binding fragments of g3p, mutants or variants thereof, orcompounds, polypeptides and fusion proteins comprising g3p, amyloidbinding fragments of g3p, or mutants or variants thereof; or (e)modified filamentous phage bearing variants of g3p, amyloid bindingfragments of g3p (not as part of a displayed g3p protein), mutants orvariants of such binding fragments, or fusion proteins or otherheterologous polypeptides that comprise g3p, amyloid binding fragmentsof g3p, or mutants or variants thereof). The diagnostic agent mayfurther comprise a detectable label, or may be be otherwise detected invivo.

In some embodiments, a composition of the invention, such as onecomprising a soluble g3p or an amyloid binding fragment (includingmutants and variants thereof), or a g3p fusion protein, is used as anamyloid imaging agent. The imaging agent can detect amyloid and diagnosediseases associated with amyloid. Because the compositions of theinvention bind amyloid irrespective of the type of fiber, they areadvantageous in that they can image any amyloid aggregate (Aβ, tau,α-synuclein, etc.)—all with a single imaging agent. At present, thereare no acceptable imaging agents/methods for tau or alpha synucleinaggregates in the CNS. And while imaging agents for β-amyloid exist,there is still a need for additional agents that may provide improvedcorrelation between cognitive function and imaging results and/or thatbetter predict which patients will deteriorate versus remain stable. Fora review, see Resnick & Sojkova, Alzheimer's Res Ther. (2011) 3(1):3.

The diagnostic compositions of the invention may be used as imagingagents in combination with an imaging agent that is specific forβ-amyloid such as, for example, F18-AV-45, Eli Lilly. Since there arecurrently no known imaging agents for non-β-amyloid aggregates, the useof a diagnostic composition of the invention together with aβ-amyloid-specific imaging agent will result in the detection ofnon-β-amyloid aggregates based on differential detection. Thus, in oneembodiment, a diagnostic composition of the invention is used as animaging agent in combination with a β-amyloid imaging agent to detectnon-β-amyloid aggregates.

In another embodiment, a diagnostic composition of the invention is usedas an imaging agent to detect β-amyloid in the CNS, including the brain.

A diagnostic composition of the invention generally requires that theamyloid-binding component be attached to one or more detectable labelswhen it is used as an imaging agent. Various labels can be attached tothe amyloid binding component of the diagnostic composition usingstandard techniques for labeling proteins. Examples of labels includefluorescent labels and radiolabels. There are a wide variety ofradiolabels that can be used, but in general the label is often selectedfrom radiolabels including, but not limited to, ¹⁸F, ¹¹C, and ¹²³I.These and other radioisotopes can be attached to the protein using wellknown chemistry. In one embodiment, the label is detected using positronemission tomography (PET). However, any other suitable technique fordetection of radioisotopes may also be used to detect the radiotracer.

Diagnostic compositions of the invention may be administered using thesame routes described for therapeutic compositions. In one embodiment,intrathecal administration is used as the route for administering thediagnostic composition. In another embodiment, intravenousadministration is used as the route for administering the diagnosticcomposition.

EXAMPLES

Although the demonstrated therapeutic efficacy of filamentous phage asbinding and anti-aggregation agents is not contingent upon anyparticular mechanism of action, understanding the mechanism permits thedesign of phage with greater therapeutic efficacy. In addition, itserves as a basis for preparing additional anti-aggregation agents.

As noted previously, M13 has been shown to bind to and disaggregate atleast four different amyloid fibers: amyloid-β 1-42 fibers (fAβ42),α-synuclein fibers (fαsyn), yeast prion NM fibers (fNM), and tau fibers(ftau). The four proteins that make up these amyloid fibers haveunrelated primary amino acid sequence, but all four are misfolded intothe canonical amyloid fold. Eichner & Radford, 2011. The ability of M13to bind to and mediate disaggregation of each of these indicates thatM13 recognizes a structural motif, such as cross-beta sheet conformationor a conformational feature such as hydrophobic groves, both of whichare defining characteristics of all amyloid fibers.

But amyloid disaggregation is not a general property of all phage. Forexample, the structurally distinct icosahedral phage T7 does not mediatedisaggregation of fAβ42, even when T7 is incubated with fAβ42 for 3 daysat 37° C.

Bacteriophage T7 did not show any dissociation activity even atconcentrations at which M13 dissociates over 70% of the co-incubatedamyloid fibers. In contrast, the bacteriophage fd, which carries anegatively charged amino acid in its g8p compared to M13 (and thereforedisplays 2800 more negative charges/phage than M13 given the copy numberof g8p), bound and disaggregated fAβ42 similar to M13. These initialstudies, along with the finding that amyloid disaggregation could alsobe mediated by tobacco mosaic virus (TMV) E. coli pili, and the tailtubes of T4, all of which also have a helical cylinder shape andrepeating units (see US 2011/0182948), suggested that it may be theshape of the phage that is critical for its amyloid fiber-disassociationactivity.

However, the following examples describe an alternate (although notmutually exclusive) mechanism for the reported binding andanti-aggregation property of filamentous phage. Based on these examplesand the mechanism of action they support, modified phage with improvedbinding to amyloid are provided along with new amyloid-binding agents.

Example 1 M13 Phage Preferentially Binds Aβ Fibrils

Binding of M13 to Aβ fibrils versus Aβ monomers was determined bysurface plasmon resonance (SPR).

M13 phage preferentially binds Aβ fibrils; it does not bind Aβ monomers.Surface plasmon resonance studies using 10¹⁴ phage/mL flowed across abiosensor chip with immobilized fAβ are reported in FIG. 3. FIG. 3 showsthat the K_(D) of M13 binding is about 4 nM, which is comparable tobinding by a monoclonal antibody. This high affinity interactionindicates that a specific binding process is occurring between phage andthe amyloid fiber.

Example 2 Binding of M13 to Ab Fibrils is Dose Dependant

M13 binding to fAβ42 is also dose dependent. In FIG. 4A, the binding oftwo phage doses with increasing molar amounts of fAβ42 was compared. Inthis M13-Amyloid fiber binding assay, M13-Alexa488 was mixed with Aβ(fAβ) for 2-3 hours to allow complexes to form, then the complexsedimented by centrifugation at 7500 rpm for 10 minutes. Thefluorescence in the pellet was proportional to the M13 bound to theamyloid. This assay provides both a quantitative measure of binding ofphage to fAβ and provides a system for assessing the ability of otheragents to compete with phage for binding. FIG. 4B shows that the K_(D)for M13 binding competition is similar to that observed for bindingusing surface plasmon resonance.

Example 3 Binding of M13 to Aβ Fibrils Requires Native Conformation

When M13 phage is heated at 90° C. for 10 minutes, its ability tocompete for binding is essentially abrogated. FIG. 5 shows bindingcompetition results using heat treated (boxes) versus nativeconformation (circles) M13 in the amyloid fiber competition bindingassay.

Example 4 Temperature Correlates with Mβ-Amyloid Interactions

M13 potently disaggregates amyloid fibers. FIG. 6 shows a Thioflavin T(ThT) fluorescence assay using fAβ. In the presence of M13, fAβ42disaggregates.

FIG. 7A shows that changing the salt concentration in the ThTfluorescence 10 fold (from 0.15 to 1.5 M) results in only a 2-3 folddifference in the percentage of fAβ that is disaggregated. Thisindicates that hydrophobic interactions are responsible for most of thedisaggregation observed.

In contrast to the relatively minor effect of salt concentration, FIG.7B shows that changing the temperature from 4° C. to 37° C. results inan 8-10 fold difference in disaggregation.

These results indicate that M13 disaggregation is dependent on a proteinthat is more active at a higher temperature and that is relativelyinsensitive to the effect of salt in the assay, implying a hydrophobicinteraction. Phage g3p fits this description. Its N1 and N2 domains arelinked by a flexible glycine-rich linker that “opens” up followingbinding of N2 to the bacterial F-pilus. N1 is then available for bindinga bacterial co-receptor as part of the infection process. Increasing thetemperature in the disaggregation assay is expected to “open” up the N2and N1 domains of g3p.

While inactivating M13 at high temperature (90° C., 10 minutes, see FIG.5) abrogates binding, increasing the incubation temperature in theMβ-amyloid binding assay has a positive effect on binding. FIG. 8A showsthat increasing the temperature from 18° C. to 58° C. results inprogressively better binding up to about the hinge unfolding T_(M) ofabout 50° C., at which point binding begins to decrease. This optimalbinding temperature is consistent with the temperature of the N1-N2unfolding (the so-called melting temperature, or T_(M)) in g3p, which is48.1° C. Increasing the incubation temperature to 50° C. vs 37° C. alsoresults in more rapid binding of M13 to fAβ42. FIG. 8B.

Example 5 g3p is Required for M13-β-Amyloid Interaction

To directly test whether g3p is required for M13-β-amyloid interaction,g3p was removed from phage by proteolytic treatment with ArgC (M13Δg3p)and the M13Δg3p phage compared to refolded phage for Aβ binding.Treatment with ArgC, a Bacillus protease, selectively removes the g3psubunits from phage. The results are presented in FIG. 9A. Refolded M13still competes with wildtype M13 in the competition binding assay,albeit at a decreased level. However, even 15 fold of M13Δg3p competedpoorly, if at all with wildtype M13. This inability to compete with wildtype M13 is consistent with a loss of infectivity in the M13Δg3p phage.FIG. 9B. ArgC treatment also caused a loss of disaggregation activity.FIG. 9C.

If g3p is mediating binding in a manner analogous to its role ininfection, then the N1 and N2 domains that are important for infectionshould also compete with M13 for binding. To test this, recombinantsoluble N1N2 (“rs-g3p(N1N2)”; “Construct 3”) was prepared and tested inthe competition assay. As shown in FIGS. 10A and 10B, M13 competes withthe labeled M13 for binding to fAβ42, but M13Δg3p does not. In contrast,rs-g3p(N1N2) was able to compete with M13, indicating that the N1 and N2domains of g3p are sufficient for β-amyloid binding. Similar resultswere obtained in a repeat of the competition assay. FIG. 10B.

Example 6 g3p Hinge Unfolding Mutations Modulate Amyloid Binding

Mutations that affect the ability of the hinge between the N1 and N2domains of g3p to open up should also affect the ability of phagebearing those mutations to compete with wildtype M13 for binding to Aβ.Eckert & Schmid, 2007, described several variant phage that were used totest this hypothesis. Variant “AAA” (also known as “3A”) impairs pilusbinding and decreases the stability of the N2 domain. AAA carries thefollowing mutations in g3p: W181A, F190A, and F194A. IIHY contains themutations T13I, T101I, Q129H, and D209Y, which stabilize the N2 domainand increase T_(M).

Binding competition was assessed for phage fd, which has the same aminoacid sequence as M13 g3p in the N1 and N2 domains (FIG. 2); IIHY, whichhas a higher hinge Tm than M13, and AAA. Phages fd, AAA, and IIHY werepre-activated at 50° C. for 1.5 hours, then activated and non-activatedFd, AAA, & IIHY were compared for their ability to compete with labeledM13. FIG. 11 presents the results. Wild type fd was a better competitorwhen activated by heating. In contrast, heating had little effect onIIHY, which has a higher hinge Tm. AAA, which has decreased N2 domainstability relative to M13, was a better competitor with or without heatpretreatment.

These data support the conclusion that the interaction of M13 withβ-amyloid is via a mechanism similar to that by which M13 infectsbacteria. First, they indicate that hydrophobic interactions areimportant for the M13-β-amyloid interaction. Second, the temperaturedependence of M13 binding and disaggregation activities reflect theN1-N2 hinge unfolding Tm. Third, selective proteolysis of g3p abrogatesM13-β-amyloid interactions.

Example 7 A g3p Fragment Selectively & Potently Binds Amyloid, but notMonomers

To assess whether a g3p fragment retains the ability to bind to amyloid,a g3p fragment comprising N1 and N2 was prepared and assessed for itsability to bind Aβ fibrils versus Aβ monomers by surface plasmonresonance (SPR). The results indicate that rs-g3p(N1N2) preferentiallybinds Aβ fibrils; it does not bind Aβ monomers. Surface plasmonresonance studies using 4 μM rs-g3p(N1N2) are reported in FIG. 13, whichalso shows the K_(D) of rs-g3p(N1N2) binding to be about 160 nM. Thishigh affinity interaction indicates that a specific binding process isoccurring between rs-g3p(N1N2) and the amyloid fiber.

Additional constructs were assessed by SPR. The table below summarizesthe results.

ka Analytes (1/M · s) kd (1/s) K_(D) Construct 1  2.6e3  9.2e−6 3.59 nMM13 Construct 3  1.5e3  2.4e−4 0.15 uM rs-G3P (N1N2), 25° C. Construct 3 4.1e3   2e−4 0.05 uM rs-G3P (N1N2), preheated at 37° C. Construct 41.75e4 1.28e−4 7.32 nM rs-g3p (N1N2)-hIgG4Fc fusion protein, 25° C.Construct 5 1.52e4 1.66e−4 10.9 nM rs-g3p (N1N2)-hIgG4Fc fusion protein,25° C. Construct 6 1.71e4 1.58e−4  9.2 nM N1N2-IgG1Fc fusion protein,25° C.

Example 8 A g3p Fragment Potently Disaggregates Aβ42 Fibers

To test whether a g3p fragment can disaggregate amyloid fibers,rs-g3p(N1N2) was tested in a ThT fluorescence assay for its ability todegrade preformed fAβ42 fibrils. The results indicate that rs-g3p(N1N2)potently disaggregates fAβ42. FIG. 14A shows the results of thisexperiment, that rs-g3p(N1N2) disaggregates fAβ42 in a dose dependentfashion. FIG. 14B shows the IC₅₀ to be approximately 20 nM.

In a separate experiment, Aβ42 was incubated with or withoutrs-g3p(N1N2) at a concentration of 2 μM for seven days at 37° C. and theintegrity of the Aβ42 fibers was assessed by transmission electronmicrograpy. FIG. 15A shows the results of this experiment, thatrs-g3p(N1N2) disaggregates Aβ42 fibers. FIG. 15B reports the results ofa ThT assay on these same samples. Rs-g3p(N1N2) degraded preformed Aβ42fibers in this ThT assay.

Example 9 rs-g3p(N1N2) Blocks α-Synuclein and Aβ Assembly andrs-g3p(N1N2)-hIgG1-Fc Blocks Assembly and Inhibits Aggregation of Aβ

To determine whether g3p can block α-synuclein fiber assembly, and alsoto determine whether the valency (i.e., the number of copies of g3p)plays a role, an assay testing the ability of pentameric g3p (5 copiesof g3p) and monomeric g3p (one copy of g3p) to block α-synucleinactivity was conducted. The results show that g3p blocks α-synucleinfiber assembly, and that pentameric g3p is more efficient than monomericg3p at this activity. See FIG. 16.

The ability of rs-g3p(N1N2) (Construct 3) and rs-g3p(N1N2)-hIgG1-Fc(Construct 6) to inhibit assembly of Aβ42 was also assessed. As shown inFIG. 30 and FIG. 31, Construct 3 and Construct 6 are capable ofinhibiting the assembly of fAβ42 in a dose-dependent fashion. As shownin FIG. 37, Construct 3 and Construct 6 are capable of inhibiting fAβ42aggregation.

Example 10 rs-g3p(N1N2)-Ig Fusion Protein Binds to and Disaggregates Aβ

To assess whether g3p valency plays a role in the potency of g3p bindingto amyloid, an Ig fusion protein that is bivalent for rs-g3p(N1N2)(“rs-g3p(N1N2)-Ig fusion”) was made and compared with pentavalent M13for its ability to bind to Aβ fibers. As shown in FIG. 17,rs-g3p(N1N2)-Ig fusion binds to Aβ with similar affinity as M13, andmore potently than rs-g3p(N1N2) alone, indicating that the valency ofg3p may be important. Similar results were obtained in a repeat of thecompetition assay. FIG. 18. In FIG. 18, the squares represent Construct2 (M13); the triangles represent Construct 3 (rs-g3p(N1N2)); the upsidedown triangles represent Construct 4 (rs-g3p(N1N2)-Ig fusion); and thediamonds represent a r-IgG4 Fc negative control.

To assess whether or not valency also plays a role in disaggregation,bivalent rs-g3p(N1N2)-Ig fusion (“Construct 4”) was compared topentavalent M13 in a filter trap assay. FIG. 19. The results indicatethat both bivalent rs-g3p(N1N2)-Ig fusion and pentavalent M13 potentlydisaggregate β-amyloid fibers. Also indicated is that valency may beimportant for potency of disaggregation, as indicated by the ability of1.7 nM pentavalent M13 to reduce aggregates at a level similar to 40 nMrs-g3p(N1N2)-Ig fusion. FIG. 19.

In a similar assay, 1×10¹²/ml M13 (Construct 2); 80 nm and 800 nMrs-g3p(N1N2)-hIgG4-Fc (Construct 5); and 80 nm and 800 nM ofrs-g3p(N1N2)-hIgG1-Fc (Construct 6) were assayed for their ability todisaggregate Aβ42 fibers in a filter trap assay. Constructs 2, 5, and 6potently disaggregate β-amyloid fibers. FIG. 33.

Example 11 Tetrameric Streptavidin-[Biotin-g3p(N1N2)] Protein Binds toand Disaggregates fAβ

To further assess the role of valency on g3p's ability to bind anddisaggregate amyloid, a tetrameric streptavidin conjugated g3p(N1N2) wasprepared by combining rs-g3p(N1N2) with Biotin-Lys-NTA in the presenceof NiSO₄. Excess ligand was removed using a MWCO 3 KDa membrane.Streptavidin was added, and excess rs-g3p(N1N2)-Biotin was removed usinga MWCO 100 KDa membrane. The resulting g3p construct,streptavidin-[biotin-g3p(N1N2)], has four rs-g3p(N1N2) moieties.Streptavidin-[biotin-g3p(N1N2)] was compared to rs-g3p(N1N2) (“Construct3”) in a binding assay. FIG. 20. Tetramericstreptavidin-[biotin-g3p(N1N2)] bound to fAβ more potently thanmonomeric rs-g3p(N1N2), providing a further indication that valency isimportant for potency of binding. FIG. 20. However, even monomericrs-g3p(N1N2) bound to fAβ in therapeutically acceptable levels.

To assess whether or not valency also plays a role in disaggregation,monomeric rs-g3p(N1N2) was compared to tetramericstreptavidin-[biotin-g3p(N1N2)] in a filter trap assay. FIG. 21. Theresults indicate that both monomeric rs-g3p(N1N2) and tetramericstreptavidin-[biotin-g3p(N1N2)] potently disaggregate fAβ fibers. Alsoindicated is that valency may be important for potency ofdisaggregation, as indicated by the superior ability of 360 nMtetrameric streptavidin-[biotin-g3p(N1N2)] to abrogate up to 200 ng fAβaggregates, as compared to the reduced disaggregation of Aβ by 2.5 μMmonomeric rs-g3p(N1N2). FIG. 21, row 2 compared to row 4, for example.

Disaggregation of Aβ by streptavidin-[biotin-g3p(N1N2)] was alsoassessed by TEM. Streptavidin-[biotin-g3p(N1N2)] completelydisaggregated fAβ42 after a three day incubation. FIG. 22.

Example 12 N1N2-Ig Fusion Protein Significantly Reduces Aβ in a MurineModel of Alzheimers Disease

Using a well known mouse model for studying Alzheimer's Disease (Hsiaoet al., Science (1996) 274:99-102; Duyckaerts et al., Acta Neuropathol(2008) 115:5-38), male Tg2576 mice were aged to greater than 500 days,injected (2 μL/injection) bilaterally into the hippocampus with twodifferent preparations of N1N2-Ig fusions (Construct 5 at 7.8μg/injection and Construct 6 at 8.2 μg/injection) or saline as anegative control, and sacrificed on day 7. Brain tissue was harvested,sectioned, and stained for plaque load quantification using ananti-amyloid beta monoclonal antibody (82E1; cat. # MBS490005-IJ10323from MyBioSource). As shown in FIG. 28, both N1N2-Ig fusion proteinssignificantly reduced the plaque load measured in the hippocampuscompared to saline-treated mice. As shown in FIG. 29, both N1N2-Igfusion proteins significantly reduced the plaque load measured in thecerebral cortex compared to saline-treated mice.

Example 13 N1N2-Ig Fusion Protein Blocks Aβ Oligomer InducedCytotoxicity

Aβ oligomers cause the release of certain toxic enzymes in neuronalcells. The enzyme can be assayed to determine whether a compound caninhibit the Aβ oligomer induced cytotoxicity. FIG. 32 presentsrepresentative data showing that M13 (Construct 2) andrs-g3p(N1N2)-hIgG1-Fc (Construct 6) block oligomer-induced toxicity toN2a cells. g3p fragments are therefore potent inhibitors of Aβ oligomerinduced cytotoxicity.

Example 14 N1N2-Ig Fusion Protein Binds and Disaggregates Tau

To assess whether a g3p fragment binds to tau, a g3p fragment-Ig fusionprotein comprising N1 and N2 was prepared and assessed for its abilityto bind ftau by surface plasmon resonance (SPR). FIG. 35 shows theresults of one representative SPR assay showing thatrs-g3p(N1N2)-hIgG4-Fc (Construct 4) potently binds ftau.

To test whether a g3p fragment can disaggregate tau, a g3p fragment-Igfusion protein comprising N1 and N2 was tested in a ThT fluorescenceassay for its ability to degrade preformed ftau. The results indicatethat an N1N2-Ig fusion protein potently disaggregates ftau. See, FIG.36A and FIG. 36B.

Example 15 N1N2-Ig Fusion Protein Inhibits PrP^(Sc) Accumulation,Aggregation and PrP^(Sc) Formation in a Cell Culture Model of PrionDisease (N2a22L^(Sc))

Prion diseases are characterized by the conversion of normal cellularprion protein (PrP^(c)) to the protease-resistant pathological formPrP^(Sc). PrP^(Sc) is distinguished from PrP^(c) on the basis ofprotease resistance: protease partly degrades PrP^(Sc) to form aprotease-resistant C-terminal core fragment (PrPres), which has anunglycosylated form with a molecular weight of 19-21 kDa. Inhibition,reversal, and reduction of PrP^(Sc) constitutes a viable therapeuticapproach to treatment of several degenerative diseases.

To determine whether a g3p fragment-Ig fusion protein comprising N1 andN2 (Construct 6) interferes with the formation of pathological prionconformers (PrP^(Sc)) in in vitro models of prion disease, and to verifydisaggregation or change in solubility of PrP in N2a22L^(Sc) cells inthe presence or absence of Construct 6, cells were cultured for 24 h inthe absence or presence of 1 ug/ml Construct 6 or IgG and harvested inlysis buffer. 100 μg of total protein was ultracentrifuged at 4° C. for90 min at 55,000 rpm in a TLA 100.1 rotor in a Beckman Optima TLultracentrifuge. 25 μl samples of solubilized pellets and supernatantswere subjected to SDS-PAGE and downstream analysis with anti-PrPantibody 6D11 mAb. Increased detergent insolubility precedes acquisitionof proteinase K (PK) resistance by PrP^(Sc) or PrP mutants, thereforethe ability of Construct 6 to alter PrP solubility was assessed.Construct 6-treated cells exhibited significantly reduced amounts ofaggregated/insoluble PrP compared to IgG treated N2a22L^(Sc) cells. SeeFIG. 38A and FIG. 38B.

For FIGS. 38A and 38B, N2a22L^(Sc) cells were be generated as describedpreviously (Pankiewicz et al., Eur. J. Neurosci. (2006) 23:2635-2647.Briefly, brains of terminally sick CD-1 mice infected with mouse-adapted22L prion strain were homogenized by sonication (10% weight/volume) incold phosphate-buffered saline and 5% dextrose in sterile conditions.For infection, the brain homogenate was further diluted to 2% inOpti-MEM and added to subconfluent six-well plates (Corning, Acton,Mass., USA), 1 mL per 10-cm² well. After 5 h, 1 mL of regular MEM wasadded and the cells were incubated in the presence of infectious brainhomogenate for an additional 12 h. The cells are washed and standard MEMgrowth media is added. Cells were grown until confluent and then splitinto 1:2 dilutions and transferred to 25-cm² flasks (Corning). Cellsgrown in one of the flasks were split 1:2 every 4 days to give rise tosubsequent passages, whereas cells grown in the other flask wereharvested and homogenized to monitor the level of PrP^(Sc). Based onprior studies, the presence of inoculum derived PrP^(Sc) is onlydetected in the first and second passages, so passage 4 (P4) cells wereutilized for all subsequent studies. Cells were lysed in a homogenizingbuffer composed of (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM ethyleneglycol tetraacetic acid (EGTA), 1 mM Na₃VO₄, 1 mM NaF, 2.5 mM Na₄P₂O₇, 1mM β-glycerophosphate, 1% NP-40, 0.25% sodium deoxycholate, 0.5 mMphenylmethylsulfonylfluoride (PMSF), 1 mM leupeptin, 1 mM pepstatin A, 1mM) or without PMSF for PK digestion) for 5 min at 4° C. and insolublematerials were removed by centrifugation at 10,000 g for 10 min at 4° C.For cellular fractionation, 100 μg of protein was spun at 55,000 rpm for90 min, after which the pellet was reconstituted in the starting volume.20% of both pellet and supernatant were resolved and characterizedbiochemically.

To address whether Construct 6 dose-dependently alters the propagationof PrP^(Sc), by disaggregation or changing its physicochemicalproperties, N2a22L^(Sc) cells were cultured for 24 h in the absence orpresence of increasing concentrations of Construct 6 or IgG andharvested in lysis buffer. Aliquots of lysed cells with and without PKtreatment were subjected to SDS-PAGE and downstream analysis withanti-PrP antibody 6D11 and 6H4 mAb. PrP immunoreactivity inbiochemically resolved PK digested and undigested lysate from ControlIgG and Construct 6-treated cells was assessed. Treatments included:N2a22L^(Sc)+10 μg/ml, 3 μg/ml, 1 μg/ml, 0.333 μg/ml, 0.111 μg/ml, 0.037μg/ml, 0.012 μg/ml, and 0.004 μg/ml Construct 6, or N2a22L^(Sc)+1 μg/mlmIgG.

The results indicate a significant dose-dependent decrease in PrP^(Sc)in the presence of Construct 6, with 50% less PrP^(Sc) generated in thepresence of 0.08 ug/ml Construct 6 compared to 1 ug/ml IgG. See FIG. 39Aand FIG. 39B. Repeated experiments confirmed these results.

To assess proteinase K (PK)-resistant conformer of PrP, aliquots oflysed cells, were treated with PK (1 μg/μg) 1:50 dilution at 37° C. for30 min, according to previous methods (Perrier et al., J. Neurochem(2004) 84:454-463, Pankiewicz et al., 2006). After incubation, digestionis stopped by the addition of PMSF to 4 mm.

Protein concentrations were determined using the BCA protein assay kit(Pierce). Samples were diluted in sample buffer (250 mM Tris-HCl, pH6.8, 10% SDS, 5 mM 3-mercaptoethanol, 50% glycerol, 0.02% coomassie blueG250) and boiled for 5 min. Processed samples were resolved by SDS-PAGEunder reducing conditions.

Anti-PrP monoclonal antibody 6D11 (See Sadowski et al., Neurobiol Dis.(2009) 34(2): 267-278) and 6H4 (See Cordes et al., J Immunol Methods(2008) 337:106-120) as well as anti-actin were used to characterize thesamples. The antigen-antibody complexes were detected using horseradishperoxidase-conjugated anti-mouse IgG (GE Healthcare UK Limited,Buckinghamshire, UK) and visualized using the ECL system (GE HealthcareUK Limited) following the manufacturer's instructions. Quantification ofprotein bands was performed by densitometric analysis of the films(Image J, NIH).

Taken together, the results shown in FIGS. 38A and 38B and FIGS. 39A and39B demonstrate the ability of a g3p fragment Ig fusion protein todirectly inhibit PrP^(Sc) formation in vitro.

1-59. (canceled)
 60. A method of treating a subject for a disease ordisorder associated with misfolded and/or aggregated amyloid protein,comprising administering to the subject a polypeptide, wherein thepolypeptide comprises an amyloid-binding domain selected from at leastone of: (a) a wild-type g3p, (b) an amyloid-binding fragment ofwild-type g3p, or (c) a mutant of (a) or (b) that retains the ability tobind to amyloid; and wherein the subject is not administered afilamentous bacteriophage.
 61. A method of treating a subject for adisease or disorder associated with misfolded and/or aggregated amyloidprotein, comprising administering to the subject a fusion protein,wherein the fusion protein comprises an amyloid-binding domain and atleast one additional protein or protein domain with which theamyloid-binding domain is not normally associated, wherein theamyloid-binding domain comprises at least one of: (a) a wild-type g3p,(b) an amyloid-binding fragment of wild-type g3p, or (c) a mutant of (a)or (b) that retains the ability to bind to amyloid.
 62. The method ofclaim 2, wherein the at least one additional protein or protein domaincomprises an immunoglobulin constant region.
 63. The method of claim 3,wherein the immunoglobulin constant region is an immunoglobulin Fcfragment.
 64. The method of claim 4, wherein the immunoglobulin Fefragment is an Fe fragment of IgG or IgM.
 65. The method of claim 5,wherein the IgG is IgG1.
 66. The method of claim 1, wherein theamyloid-binding portion comprises an amyloid-binding fragment of the N2domain of wild-type g3p or a corresponding fragment of a mutant g3p. 67.The method of claim 1, wherein the wild-type g3p or amyloid-bindingfragment of wild-type g3p is identical to the amino acid sequence of thecorresponding portion of SEQ ID NO:1.
 68. The method of claim 1, whereinthe polypeptide is at least 85% identical to the amino acid sequence ofthe corresponding portion of SEQ ID NO:1.
 69. The method of claim 1,wherein the polypeptide is at least 90% identical to the amino acidsequence of the corresponding portion of SEQ ID NO:1.
 70. The method ofclaim 1, wherein the polypeptide is at least 95% identical to the aminoacid sequence of the corresponding portion of SEQ ID NO:1.
 71. Themethod of claim 1, wherein the polypeptide is at least 98% identical tothe amino acid sequence of the corresponding portion of SEQ ID NO:1. 72.The method of claim 1, wherein the polypeptide comprises an amino acidsequence that is at least 95% identical to the N1-N2 fragment of SEQ IDNO:1.
 73. The method of claim 2, wherein the amyloid-binding polypeptidecomprises an amino acid sequence that is at least 95% identical to theN1-N2 fragment of SEQ ID NO:1.
 74. The method of claim 1, wherein theamyloid-binding polypeptide comprises an N1-N2 fragment of wild-type g3por a corresponding fragment of a mutant g3p.
 75. The method of claim 2,wherein the amyloid-binding polypeptide comprises an N1-N2 fragment ofwild-type g3p or a corresponding fragment of a mutant g3p.
 76. Themethod of claim 1, wherein the amyloid-binding polypeptide compriseswild-type or mutant full-length g3p.
 77. The method of claim 1, whereinthe amyloid-binding polypeptide comprises an N1-N2 fragment of g3p,wherein the hinge region of N2 is mutated to result in a polypeptidewith reduced hinge melting temperature and higher affinity for amyloidas compared to a corresponding wild type M13 phage polypeptide.
 78. Themethod of claim 1, wherein the disease or disorder is selected from thegroup consisting of Alzheimer's disease, early onset Alzheimer'sdisease, late onset Alzheimer's disease, presymptomatic Alzheimer'sdisease, Parkinson's disease, SAA amyloidosis, cystatin C, hereditaryIcelandic syndrome, senility, multiple myeloma; prion diseases, kuru,Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussier-Scheinker disease(GSS), fatal familial insomnia (FFI), scrapie, bovine spongiformencephalitis (BSE), amyotrophic lateral sclerosis (ALS), spinocerebellarataxia (SCA1, SCA3, SCA6, or SCA7), Huntington disease,dentatorubral-pallidoluysian atrophy, spinal and bulbar muscularatrophy, hereditary cerebral amyloid angiopathy, familial amyloidosis,frontotemporal lobe dementia, frontotemporal lobar degeneration (FTLD),peripheral amyloidosis, British/Danish dementia, and familialencephalopathy.
 79. The method of claim 2, wherein the disease ordisorder is selected from the group consisting of Alzheimer's disease,early onset Alzheimer's disease, late onset Alzheimer's disease,presymptomatic Alzheimer's disease, Parkinson's disease, SAAamyloidosis, cystatin C, hereditary Icelandic syndrome, senility,multiple myeloma; prion diseases, kuru, Creutzfeldt-Jakob disease (CJD),Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia(FFI), scrapie, bovine spongiform encephalitis (BSE), amyotrophiclateral sclerosis (ALS), spinocerebellar ataxia (SCA1, SCA3, SCA6, orSCA7), Huntington disease, dentatorubral-pallidoluysian atrophy, spinaland bulbar muscular atrophy, hereditary cerebral amyloid angiopathy,familial amyloidosis, frontotemporal lobe dementia, frontotemporal lobardegeneration (FTLD), peripheral amyloidosis, British/Danish dementia,and familial encephalopathy.
 80. The method of claim 19, wherein thedisease or condition is selected from the group consisting ofParkinson's disease, Alzheimer's disease, and Huntington's disease. 81.The method of claim 20, wherein the disease or condition is selectedfrom the group consisting of Parkinson's disease, Alzheimer's disease,and Huntington's disease.
 82. The method of claim 21, wherein thedisease or condition is Alzheimer's disease.
 83. The method of claim 22,wherein the disease or condition is Alzheimer's disease.
 84. The methodof claim 1, wherein the subject is positive for the biomarkerflorbetapir when that biomarker is used as an imaging agent in positronemission tomography.
 85. The method of claim 2, wherein the subject ispositive for the biomarker florbetapir when that biomarker is used as animaging agent in positron emission tomography.
 86. The method of claim1, wherein the amyloid-binding polypeptide is capable of reducingamyloid, inhibiting amyloid formation, inhibiting amyloid aggregation,or removing toxic oligomer formation, or preventing toxic oligomerformation.
 87. The method of claim 2, wherein the amyloid-bindingpolypeptide is capable of reducing amyloid, inhibiting amyloidformation, inhibiting amyloid aggregation, or removing toxic oligomerformation, or preventing toxic oligomer formation.